RPTP is essential for NCAM-mediated p59fyn activation and neurite elongation
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《联合生物学》
1 Zentrum für Molekulare Neurobiologie, Universitt Hamburg, 20246 Hamburg, Germany
2 Hubrecht Laboratory, Netherlands Institute for Developmental Biology, 3584 CT Utrecht, Netherlands
The neural cell adhesion molecule (NCAM) forms a complex with p59fyn kinase and activates it via a mechanism that has remained unknown. We show that the NCAM140 isoform directly interacts with the intracellular domain of the receptor-like protein tyrosine phosphatase RPTP, a known activator of p59fyn. Whereas this direct interaction is Ca2+ independent, formation of the complex is enhanced by Ca2+-dependent spectrin cytoskeleton–mediated cross-linking of NCAM and RPTP in response to NCAM activation and is accompanied by redistribution of the complex to lipid rafts. Association between NCAM and p59fyn is lost in RPTP-deficient brains and is disrupted by dominant-negative RPTP mutants, demonstrating that RPTP is a link between NCAM and p59fyn. NCAM-mediated p59fyn activation is abolished in RPTP-deficient neurons, and disruption of the NCAM–p59fyn complex in RPTP-deficient neurons or with dominant-negative RPTP mutants blocks NCAM-dependent neurite outgrowth, implicating RPTP as a major phosphatase involved in NCAM-mediated signaling.
V. Bodrikov, I. Leshchyns'ka, and V. Sytnyk contributed equally to this paper.
Abbreviations used in this paper: FGFR, FGF receptor; NCAM, neural cell adhesion molecule.
Introduction
The neural cell adhesion molecule (NCAM) is involved in several morphogenetic events, such as neuronal migration and differentiation, neurite outgrowth, and axon fasciculation. NCAM-induced morphogenetic effects depend on activation of Src family tyrosine kinases and, in particular, p59fyn kinase (Schmid et al., 1999). NCAM-dependent neurite outgrowth is impaired in neurons from p59fyn-deficient mice (Beggs et al., 1994) and is abolished by inhibitors of Src kinase family members (Crossin and Krushel, 2000; Kolkova et al., 2000; Cavallaro et al., 2001). The NCAM140 isoform has been observed in a complex with p59fyn, whereas p59fyn does not associate significantly with NCAM180 or glycosylphosphatidylinositol-linked NCAM120 (Beggs et al., 1997). However in oligodendrocytes, p59fyn is also associated with NCAM120 in isolated lipid rafts (Kramer et al., 1999), whereas in tumor cells NCAM is also associated with pp60c-src (Cavallaro et al., 2001), suggesting that additional molecular mechanisms may define NCAM's specificity of interactions with Src kinase family members. Several lines of evidence suggest that NCAM's association with lipid rafts is critical for p59fyn activation. NCAM not only colocalizes with p59fyn in lipid rafts (He and Meiri, 2002) but disruption of NCAM140 association with lipid rafts either by mutation of NCAM140 palmitoylation sites or by lipid raft destruction attenuates activation of the p59fyn kinase pathway, completely blocking neurite outgrowth (Niethammer et al., 2002). However, in spite of compelling evidence that NCAM can activate Src family tyrosine kinases, the mechanism of this activation has remained unclear.
The activity of Src family tyrosine kinases is regulated by phosphorylation (Brown and Cooper, 1996; Thomas and Brugge, 1997; Bhandari et al., 1998; Hubbard, 1999; Petrone and Sap, 2000). The two best-characterized tyrosine phosphorylation sites in Src family tyrosine kinases perform opposing regulatory functions. The site within the enzyme's activation loop (Tyr-420 in p59fyn) undergoes autophosphorylation, which is crucial for achieving full kinase activity. In contrast, phosphorylation of the COOH-terminal site (Tyr-531 in p59fyn) inhibits kinase activity through intramolecular interaction between phosphorylated Tyr-531 and the SH2 domain in p59fyn, which stabilizes a noncatalytic conformation.
A well known activator of Src family tyrosine kinases is the receptor protein tyrosine phosphatase RPTP (Zheng et al., 1992, 2000; den Hertog et al., 1993; Su et al., 1996; Ponniah et al., 1999). It contains two cytoplasmic catalytic domains, D1 and D2, of which only D1 is significantly active in vitro and in vivo (Wang and Pallen, 1991; den Hertog et al., 1993; Wu et al., 1997; Harder et al., 1998). To activate Src family tyrosine kinase, constitutively phosphorylated pTyr789 at the COOH-terminal of RPTP binds the SH2 domain of Src family tyrosine kinase that disrupts the intra-molecular association between the SH2 and SH1 domains of the kinase. This initial binding is followed by binding between the inhibitory COOH-terminal phosphorylation site of the Src family tyrosine kinase (pTyr531 in p59fyn) and the D1 domain of RPTP resulting in dephosphorylation of the inhibitory COOH-terminal phosphorylation sites in Src family tyrosine kinases (Zheng et al., 2000). These sites are hyperphosphorylated in cells lacking RPTP, and kinase activity of pp60c-src and p59fyn in RPTP-deficient mice is reduced (Ponniah et al., 1999). Like p59fyn and NCAM, RPTP is particularly abundant in the brain (Kaplan et al., 1990; Krueger et al., 1990), accumulates in growth cones (Helmke et al., 1998), and is involved in neural cell migration and neurite outgrowth (Su et al., 1996; Yang et al., 2002; Petrone et al., 2003).
Remarkably, a close homologue of RPTP, CD45, associates with the membrane-cytoskeleton linker protein spectrin (Lokeshwar and Bourguignon, 1992; Iida et al., 1994), a binding partner of NCAM (Leshchyns'ka et al., 2003). Following this lead, we investigated the possibility that RPTP is involved in NCAM-induced p59fyn activation. We show that the intracellular domains of NCAM140 and RPTP interact directly and that this interaction is enhanced by spectrin-mediated Ca2+-dependent cross-linking of NCAM and RPTP. Levels of p59fyn associated with NCAM correlate with the ability of NCAM-associated RPTP to bind to p59fyn, and the NCAM–p59fyn complex is disrupted in RPTP-deficient brains implicating RPTP as linker molecule between NCAM and p59fyn. RPTP redistributes to lipid rafts in response to NCAM activation and RPTP levels are reduced in lipid rafts from NCAM-deficient mice, suggesting that NCAM recruits RPTP to lipid rafts to activate p59fyn. Finally, NCAM-mediated p59fyn activation is abolished in RPTP-deficient neurons and NCAM-induced neurite outgrowth is blocked in RPTP-deficient neurons or neurons transfected with dominant-negative RPTP mutants, demonstrating that RPTP is a major phosphatase involved in NCAM-mediated signaling.
Results
Activation of p59fyn is impaired in NCAM-deficient brains
Cross-linking of NCAM at the cell surface results in a rapid activation of p59fyn kinase (Beggs et al., 1997; Niethammer et al., 2002) via an unknown mechanism. To analyze whether or not NCAM deficiency may affect the activation status of p59fyn, we compared levels of activated p59fyn characterized by dephosphorylation at Tyr-531 and phosphorylation at Tyr-420 in the brains of wild-type and NCAM-deficient mice. Whereas the level of p59fyn protein was higher in brain homogenates of NCAM-deficient mice (Fig. 1 A), labeling with antibodies recognizing only p59fyn dephosphorylated at Tyr-531 or with antibodies recognizing only p59fyn phosphorylated at Tyr-420 was reduced in brain homogenates of NCAM-deficient mice (Fig. 1 B), indicating that activation of p59fyn is inhibited in NCAM-deficient brains and suggesting that NCAM is involved in the regulation of p59fyn function.
NCAM forms a complex with RPTP
The intracellular domain of NCAM does not contain sequences known to induce p59fyn activation. Thus, NCAM may form a complex with a protein, possibly a protein tyrosine phosphatase, to activate p59fyn. One possible candidate is the RPTP that dephosphorylates Tyr-531 of p59fyn (Bhandari et al., 1998) and is highly enriched in neurons and growth cones (Helmke et al.,1998). Remarkably, in RPTP-deficient cells, both dephosphorylation of the COOH-terminal tyrosine residue and autophosphorylation of the tyrosine residue within the activation loop of pp60c-src is reduced (von Wichert et al., 2003), resembling the phenotype of NCAM-deficient mice. Furthermore, a close homologue of RPTP, CD45, associates with the membrane-cytoskeleton linker protein spectrin (Lokeshwar and Bourguignon, 1992; Iida et al., 1994), a binding partner of NCAM (Leshchyns'ka et al., 2003). To investigate if NCAM interacts with RPTP, we analyzed the distribution of both proteins in cultured hippocampal neurons. NCAM and RPTP partially colocalized along neurites, and both proteins accumulated in growth cones where clusters of NCAM partially overlapped with accumulations of RPTP (Fig. 2 A). To verify whether or not NCAM interacts with RPTP, we induced clustering of NCAM at the cell surface of live hippocampal neurons by incubation with antibodies against NCAM. Clustering of NCAM enhanced overlap between NCAM and RPTP localization (mean correlation between NCAM and RPTP localization being 0.3 ± 0.05 and 0.6 ± 0.03 in neurons treated with nonspecific IgG or NCAM antibodies, respectively; Fig. 2 B), indicating that RPTP partially redistributed to NCAM clusters and suggesting that NCAM and RPTP form a complex. Because antibodies against RPTP were directed against its intracellular domain, RPTP contained in intracellular organelles could have been recognized as colocalizing with NCAM that associates with intracellular organelles of trans-Golgi network origin (Sytnyk et al., 2002). Thus, the redistribution of RPTP to NCAM clusters may represent redistribution of intracellular carriers containing RPTP. To analyze whether or not NCAM associates with RPTP in the plasma membrane, we transfected neurons with RPTP containing the HA tag in the extracellular domain and induced clustering of NCAM and HA-RPTP with antibodies against NCAM and the HA tag. HA-RPTP partially redistributed to NCAM clusters (Fig. 2 C), indicating that both proteins form a complex at the cell surface.
Finally, we examined the association between NCAM and RPTP in the brain by coimmunoprecipitation. RPTP coimmunoprecipitated with NCAM from brain homogenates (Fig. 2 D), confirming that NCAM associates with RPTP. Interestingly, we found that the level of RPTP was approximately two times higher in the brain of NCAM-deficient mice when compared with wild-type mice (Fig. 2 D), indicating a functional relationship between NCAM and RPTP.
NCAM140 is the most potent RPTP-binding NCAM isoform
To identify the NCAM isoform interacting with RPTP, we expressed NCAM120, NCAM140, and NCAM180 in CHO cells and analyzed their association with RPTP by coimmunoprecipitation. CHO cells endogenously express RPTP that was detected with RPTP antibodies as a band with a molecular mass identical to RPTP detected in brain homogenates (unpublished data). Although transfected CHO cells expressed NCAM120, NCAM140, and NCAM180 in similar amounts, RPTP coimmunoprecipitated only with NCAM140 (Fig. 3 A). However, after prolonged exposure of the film we could also detect RPTP in NCAM180 immunoprecipitates (unpublished data). RPTP did not coimmunoprecipitate with NCAM120. We conclude that RPTP associates predominantly with NCAM140 and to a lesser extent with NCAM180.
Inability of NCAM120, the GPI-linked NCAM isoform without the intracellular domain, to bind RPTP suggested that the intracellular domain of NCAM is involved in the formation of a complex between NCAM and RPTP. Furthermore, the extracellular domain of NCAM (NCAM-Fc) did not bind to RPTP in brain lysates, confirming that the extracellular domain of NCAM does not bind to RPTP (unpublished data). To verify that the NCAM intracellular domain interacts directly with the intracellular domain of RPTP, we analyzed binding of the recombinant intracellular domain of RPTP to the intracellular domain of NCAM180 or NCAM140 in a pull-down assay. For comparison, the intracellular domain of CHL1, another adhesion molecule of the immunoglobulin superfamily, was used. The intracellular domain of RPTP bound to the intracellular domain of NCAM180 or NCAM140 but not to the intracellular domain of CHL1 (Fig. 3 B). Interaction between the intracellular domains of RPTP and NCAM140 was severalfold stronger than between the intracellular domains of RPTP and NCAM180 (Fig. 3 B). To confirm this finding, we examined the direct interaction between the intracellular domains of RPTP and NCAM180 or NCAM140 by ELISA. Intracellular domain of RPTP bound to the intracellular domains of NCAM180 or NCAM140 in a concentration-dependent manner, with the intracellular domain of NCAM140 binding with a higher affinity than the intracellular domain of NCAM180 (Fig. 3 C). No binding with the intracellular domain of CHL1 was observed (Fig. 3 C). We conclude that NCAM binds directly to RPTP via the intracellular domain, with NCAM140 being the most potent RPTP-binding NCAM isoform.
RPTP binds NCAM140 via the D2 domain and links NCAM140 to p59fyn
To identify the part of the intracellular domain of RPTP responsible for the interaction with NCAM140, we coexpressed, in CHO cells, NCAM140 together with the wild-type form of RPTP (wtRPTP), RPTP lacking the D2 domain (RPTPD2), or catalytically inactive form of RPTP containing a mutation within the D1 catalytic domain (RPTPC433S) and analyzed binding of NCAM140 to these RPTP mutants by coimmunoprecipitation. All transfected RPTP constructs contained the HA tag to distinguish them from endogenous RPTP. As seen for endogenous RPTP, transfected wtRPTP coimmunoprecipitated with NCAM140 (Fig. 4 A). Similar amounts of RPTPC433S coimmunoprecipitated with NCAM140, whereas RPTPD2 did not coimmunoprecipitate (Fig. 4 A), indicating that the D2 domain is required for the interaction between RPTP and NCAM140.
Remarkably, among the major NCAM isoforms, only NCAM140 forms a complex with p59fyn (Beggs et al., 1997) that we found to correlate with its ability to bind RPTP (see the previous section). RPTP directly interacts with p59fyn (Bhandari et al., 1998). Accordingly, p59fyn coimmunoprecipitated with wtRPTP from transfected CHO cells (Fig. 4 A). Approximately the same amount of p59fyn coimmunoprecipitated with RPTPD2 (Fig. 4 A), indicating that this truncated construct also binds p59fyn probably via the D1 domain. In accordance with previous reports, p59fyn showed reduced ability to bind RPTPC433S, a catalytically inactive mutant of RPTP (Fig. 4 A; Zheng et al., 2000).
To analyze the role of RPTP in NCAM140–p59fyn complex formation, we coimmunoprecipitated p59fyn with NCAM140 in the presence of RPTP mutants. In CHO cells cotransfected with NCAM140 and wtRPTP, p59fyn coimmunoprecipitated with NCAM140 (Fig. 4 A). The amount of p59fyn coimmunoprecipitated with NCAM140 was reduced in cells cotransfected with RPTPC433S (Fig. 4 A), correlating with the reduced ability of this catalytically inactive RPTP mutant to bind p59fyn (see previous paragraph; Zheng et al., 2000). When NCAM140 was cotransfected with RPTPD2, p59fyn no longer coimmunoprecipitated with NCAM140 (Fig. 4 A). Because RPTPD2 binds p59fyn (Fig. 4 A), it is conceivable that this mutant, which does not bind NCAM140, competes with endogenous RPTP for binding to p59fyn and thus inhibits NCAM140–p59fyn complex formation.
To extend this analysis to neurons, we transfected hippocampal neurons with GFP alone or cotransfected with GFP and RPTPD2 or RPTPC433S and analyzed the redistribution of p59fyn to NCAM clusters after cross-linking NCAM with NCAM antibodies (Fig. 4 B). In neurons transfected with RPTPD2 or RPTPC433S, the level of p59fyn in NCAM clusters was reduced by 30% when compared with GFP only transfected cells, suggesting that RPTPD2 or RPTPC433S inhibit NCAM–p59fyn complex formation by competing with endogenous RPTP. The combined observations indicate that NCAM140–p59fyn complex formation correlates with the ability of NCAM140-associated RPTP to bind to p59fyn, implicating RPTP as a linker between NCAM140 and p59fyn.
Association between NCAM and p59fyn and NCAM-mediated p59fyn activation are abolished in RPTP-deficient neurons
To substantiate further our finding that RPTP is a linker protein between NCAM and p59fyn, we analyzed p59fyn activation and association of p59fyn with NCAM in RPTP-deficient brains. As for NCAM-deficient brains, levels of p59fyn dephosphorylated at Tyr-531 and levels of p59fyn phosphorylated at Tyr-420 were reduced in brain homogenates of RPTP-deficient mice (Fig. 5 A), further suggesting that RPTP plays a role in NCAM-mediated p59fyn activation in the brain. To analyze the role of RPTP in the formation of the complex between NCAM and p59fyn, we immunoprecipitated NCAM from wild-type and RPTP-deficient brains and probed immunoprecipitates with antibodies against p59fyn. Whereas p59fyn coimmunoprecipitated with NCAM from wild-type brains, p59fyn did not coimmunoprecipitate with NCAM from RPTP-deficient brains (Fig. 5 B). Furthermore, when NCAM was clustered at the surface of wild-type and RPTP-deficient cultured hippocampal neurons, levels of p59fyn were significantly reduced in NCAM clusters in RPTP-deficient neurons when compared with wild-type cells (Fig. 5 C), indicating that RPTP is required for complex formation between NCAM and p59fyn.
NCAM clustering at the cell surface induces rapid p59fyn activation (Beggs et al., 1997). To analyze whether or not RPTP is required for NCAM-induced p59fyn activation, we treated live hippocampal neurons from wild-type and RPTP-deficient mice with NCAM antibodies and analyzed levels of p59fyn dephosphorylated at Tyr-531 along neurites of the stimulated neurons. Clustering of NCAM increased levels of Tyr-531–dephosphorylated p59fyn along neurites of wild-type neurons by 60% (Fig. 5 D). However, NCAM-mediated p59fyn activation was completely abolished in RPTP-deficient neurons (Fig. 5 D), demonstrating that RPTP is required for NCAM-mediated p59fyn activation.
Formation of the complex between RPTP and NCAM is enhanced by Ca2+
Coimmunoprecipitation experiments were performed either in the presence of Ca2+ or with 2 mM EDTA, a Ca2+-sequestering agent. Whereas RPTP coimmunoprecipitated with NCAM from brain homogenates under both conditions, coimmunoprecipitated complexes were reduced by 60% in the presence of EDTA (Fig. 6 A), suggesting that Ca2+ promotes formation of the NCAM–RPTP complex. These results are in accordance with findings of Zeng et al. (1999), who found that NCAM and RPTP did not coimmunoprecipitate in the presence of EDTA. To analyze if the direct interaction between NCAM and RPTP is Ca2+ dependent, we assayed binding of the intracellular domain of NCAM140 to the intracellular domain of RPTP by ELISA in the presence or absence of Ca2+ (Fig. 6 B), showing that the direct interaction is Ca2+ independent and suggesting that additional binding partners of NCAM and/or RPTP may enhance complex formation in a Ca2+-dependent manner. Spectrin, which directly interacts with the intracellular domain of NCAM (Leshchyns'ka et al., 2003) and contains a Ca2+ binding domain (De Matteis and Morrow, 2000), is one of the possible candidates. Indeed, RPTP coimmunoprecipitated with spectrin from brain homogenates (Fig. 6 C). In the presence of 2 mM EDTA, RPTP coimmunoprecipitating with spectrin was reduced by 80% (Fig. 6 C), whereas coimmunoprecipitation of NCAM with spectrin did not depend on Ca2+ (Fig. 6 C). We conclude that RPTP directly interacts with NCAM in a Ca2+-independent manner. However, formation of the complex is enhanced by Ca2+-dependent cross-linking of NCAM140 and RPTP via spectrin.
The NCAM–RPTP complex redistributes to lipid rafts after NCAM activation
Whereas p59fyn is mainly associated with lipid rafts (van't Hof and Resh, 1997; Niethammer et al., 2002; Filipp et al., 2003), only 4–8% of all RPTP molecules were found in lipid rafts of brain (unpublished data). In hippocampal neurons extracted with cold 1% Triton X-100 to isolate lipid rafts (Niethammer et al., 2002; Leshchyns'ka et al., 2003), detergent-insoluble clusters of RPTP only partially overlapped with the lipid raft marker ganglioside GM1 (Fig. 7 A), further confirming that RPTP and p59fyn are segregated at the subcellular level. Because activation of NCAM results in its redistribution to lipid rafts (Leshchyns'ka et al., 2003), it may also promote redistribution of NCAM-associated RPTP to lipid rafts and thus activate raft-associated p59fyn. To verify this hypothesis, we studied association of NCAM and RPTP with lipid rafts in hippocampal neurons activated or not activated with NCAM-Fc or NCAM antibodies. In accordance with previous results (Leshchyns'ka et al., 2003), application of NCAM-Fc or NCAM antibodies increased GM1 levels in detergent-insoluble clusters of NCAM, indicating that NCAM redistributed to lipid rafts (Fig. 7, A–C). Application of NCAM-Fc or NCAM antibodies also increased the level of RPTP in NCAM clusters, indicating that NCAM activation promoted NCAM–RPTP complex formation (Fig. 7, A–C). Furthermore, NCAM activation also increased GM1 levels in detergent-insoluble clusters of RPTP (Fig. 7 D), confirming that NCAM-associated RPTP also redistributed to lipid rafts and suggesting that NCAM recruits RPTP to lipid rafts. To further analyze this possibility, we compared levels of RPTP in lipid rafts in brains of wild-type and NCAM-deficient mice. Indeed, RPTP was reduced by 60% in lipid rafts isolated from NCAM-deficient brains (Fig. 7 E), confirming that NCAM plays a role in RPTP targeting to lipid rafts. The levels of p59fyn were increased in NCAM-deficient lipid rafts (100% and 124 ± 7.6% in wild-type and NCAM deficient rafts, respectively) probably reflecting increased levels of p59fyn in NCAM-deficient brains. Levels of GM1 were not different in lipid rafts from wild-type and NCAM-deficient brains (100% and 103.3 ± 6.8% in wild-type and NCAM-deficient rafts, respectively), showing that lipid rafts were isolated with the same efficacy from wild-type and NCAM-deficient brains (Fig. 7 E).
NCAM-mediated recruitment of RPTP to lipid rafts is enhanced by NCAM-induced FGF receptor (FGFR)–dependent increase in intracellular Ca2+
NCAM activation increases intracellular Ca2+ concentrations via a FGFR-dependent mechanism (Walsh and Doherty, 1997; Kamiguchi and Lemmon, 2000; Juliano, 2002). This increase in intracellular Ca2+ may account for the enhanced association between NCAM and RPTP after NCAM activation (Fig. 7, A–D) because of spectrin-mediated cross-linking of NCAM140 and RPTP (Fig. 6). Interestingly, NCAM activation also induces redistribution of NCAM-associated spectrin to lipid rafts (Leshchyns'ka et al., 2003). To analyze the role of FGFR and Ca2+ in the recruitment of RPTP to an NCAM complex, we estimated levels of RPTP associated with NCAM following NCAM activation in control neurons and neurons incubated with BAPTA-AM, a membrane-permeable Ca2+ chelator (Williams et al., 1992; Cavallaro et al., 2001), or a specific FGFR inhibitor (Niethammer et al., 2002; Leshchyns'ka et al., 2003). Whereas NCAM activation increased levels of RPTP and GM1 in NCAM clusters (Fig. 7 F), treatment with BAPTA-AM or FGFR inhibitor abolished recruitment of RPTP to NCAM clusters in response to NCAM activation (Fig. 7 F). In accordance with previous findings (Leshchyns'ka et al., 2003), NCAM redistribution to lipid rafts was not affected by the FGFR inhibitor or BAPTA-AM (Fig. 7 F). BAPTA-AM or FGFR inhibitor did not affect the level of RPTP associated with NCAM under nonactivated conditions (Fig. 7 F). We conclude that, whereas at resting conditions Ca2+ does not play a major role in the interaction between NCAM and RPTP, NCAM-induced FGFR-dependent elevations of intracellular Ca2+ levels strengthen the interactions between NCAM and RPTP in response to NCAM activation, most likely via spectrin (see the section Formation of the complex between RPTP and NCAM is enhanced by Ca2+).
NCAM-induced neurite outgrowth depends on NCAM association with RPTP
NCAM-induced neurite outgrowth depends on p59fyn activation (Kolkova et al., 2000), suggesting that NCAM association with RPTP may be involved. To analyze the role of protein tyrosine phosphatases in NCAM-induced neurite outgrowth, we incubated cultured hippocampal neurons with 100 μM vanadate, an inhibitor of these phosphatases (Helmke et al., 1998). NCAM-Fc–enhanced neurite outgrowth was abolished by vanadate, indicating that activation of protein tyrosine phosphatases is required for NCAM-mediated neurite outgrowth. Vanadate did not affect neurite outgrowth in nonstimulated neurons, indicating that vanadate does not lead to nonspecific impairments (Fig. 8 A). To directly assess the role of RPTP in NCAM-induced neurite outgrowth, we transfected hippocampal neurons with the dominant-negative mutants of RPTP. Both, RPTPD2, which does not bind NCAM but associates with p59fyn, and catalytically inactive RPTPC433S, which associates with NCAM but binds p59fyn with a lower efficiency than endogenous RPTP, inhibited association of NCAM with p59fyn by competing with endogenous RPTP (Fig. 4). In neurons transfected with GFP only, stimulation with NCAM-Fc significantly enhanced neurite length when compared with control nonstimulated neurons (Fig. 8 B). However, neurons transfected with RPTPD2 or RPTPC433S remained unresponsive to NCAM-Fc stimulation (Fig. 8 B), indicating that RPTP plays a major role in NCAM-induced neurite outgrowth. To confirm this finding, we analyzed NCAM-mediated neurite outgrowth in hippocampal neurons from RPTP-deficient mice. Whereas NCAM-Fc enhanced neurite outgrowth in neurons from RPTP wild-type littermates by 100%, NCAM-Fc–induced neurite outgrowth was completely abolished in RPTP-deficient neurons (Fig. 8 C), further confirming that RPTP is required for NCAM-mediated neurite outgrowth.
Discussion
It is by now well established that in response to homophilic or heterophilic binding cell adhesion molecules of the immunoglobulin superfamily, such as NCAM, L1, or CHL1, activate Src family tyrosine kinases, and in particular pp60c-src or p59fyn, resulting in morphogenetic events, such as cell migration and neurite outgrowth. However, the mechanisms of Src family tyrosine kinase activation in these paradigms have remained unresolved. Here, we identify a cognate activator of p59fyn, the receptor protein tyrosine phosphatase RPTP, as a novel binding partner of NCAM. Activation of p59fyn is reduced in NCAM-deficient mice and interaction between NCAM and p59fyn is abolished in RPTP-deficient brains. Interestingly, we found that the levels of p59fyn and RPTP are increased in NCAM-deficient brains, possibly reflecting a compensatory reaction to the decreased activity of these enzymes in the mutant and further indicating a tight functional relationship between NCAM, RPTP, and p59fyn. NCAM-induced neurite outgrowth is completely abrogated in RPTP-deficient neurons or in neurons transfected with dominant-negative RPTP mutants, indicating that RPTP links NCAM to p59fyn both physically and functionally.
Role of Ca2+ in NCAM–RPTP–p59fyn complex formation
Interactions between NCAM and RPTP and NCAM–RPTP–p59fyn complex formation leading to neurite outgrowth are tightly regulated (Fig. 9). First, whereas direct interaction between NCAM and RPTP is Ca2+ independent, NCAM–RPTP complex formation is enhanced by Ca2+-dependent cross-linking via spectrin. Remarkably, NCAM activation results in an increase in intracellular Ca2+ concentration via influx through Ca2+ channels or release from intracellular stores, and may thus provide a positive feedback loop between NCAM activation and NCAM–RPTP complex formation involving spectrin. RPTP binding to spectrin may also elevate RPTP enzymatic dephosphorylation activity (Lokeshwar and Bourguignon, 1992). Interestingly, NCAM activation also induces activation of PKC (Kolkova et al., 2000; Leshchyns'ka et al., 2003), which is known to phosphorylate RPTP and stimulate its activity (den Hertog et al., 1995; Tracy et al., 1995; Zheng et al., 2002). Thus, a network of activated intracellular signaling molecules may underlie the induction and maintenance of NCAM-mediated neurite outgrowth. It is interesting in this respect that the NCAM140 isoform predominates in these interactions: it interacts more efficiently with p59fyn via RPTP and enhances neurite outgrowth more vigorously than NCAM180 (Niethammer et al., 2002). The structural dispositions of NCAM140 for this preference will remain to be established.
The role of lipid rafts
Additional regulation of RPTP-mediated p59fyn activation is achieved by segregation of RPTP and p59fyn to different subdomains in the plasma membrane (Fig. 9). Approximately 90% of all RPTP molecules in the brain are located in a lipid raft-free environment and are thereby segregated from lipid raft-associated p59fyn under nonstimulated conditions. Whereas segregation of receptor protein tyrosine phosphatases from their potential substrates due to targeting to different plasma membrane domains has been suggested as a general mechanism of the regulation of receptor protein tyrosine phosphatase function (Petrone and Sap, 2000), the mechanisms that target receptor protein tyrosine phosphatases to lipid rafts have remained unclear. We show that levels of raft-associated RPTP in the NCAM-deficient brain are reduced, and NCAM redistribution to lipid rafts in response to NCAM activation also induces redistribution of RPTP to lipid rafts via its NCAM association. The combined observations indicate that NCAM plays a role in recruiting NCAM-associated RPTP to lipid rafts via NCAM palmitoylation (Niethammer et al., 2002; Fig. 9) or via NCAM interaction with GPI-anchored components of lipid rafts, such as the GPI-linked GDNF receptor (Paratcha et al., 2003). Investigations on the regulatory mechanisms underlying palmitoylation will be required to understand the subcellular compartment-specific distribution of the NCAM–RPTP–p59fyn complex. Furthermore, the localization of adhesion molecules and receptors will have to be elucidated in view of lipid rafts heterogeneities.
Potential role of protein tyrosine phosphatases in signaling mediated by other cell adhesion molecules
Besides NCAM, activation of L1 and CHL1, other cell adhesion molecules of the immunoglobulin superfamily, also results in the activation of Src family tyrosine kinases (Schmid et al., 2000; Buhusi et al., 2003). As for NCAM, the intracellular domains of L1 and CHL1 do not possess structural motif for protein tyrosine phosphatase activity, suggesting that yet unidentified protein tyrosine phosphatases may be involved. Identification of protein tyrosine phosphatases associated with other cell adhesion molecules of the immunoglobulin superfamily and conjunctions with RPTP-activated integrins (Zeng et al., 2003) will be an important next step in the elucidation of the mechanisms that cell adhesion molecules use differentially to guide cell migration and neurite outgrowth in the developing nervous system.
Materials and methods
Antibodies and toxins
Rabbit polyclonal antibodies against NCAM (Niethammer et al., 2002) were used in immunoprecipitation, immunoblotting, and immunocytochemical experiments; and rat mAbs H28 against mouse NCAM (Gennarini et al., 1984) were used in immunocytochemical experiments. Both antibodies recognize the extracellular domain of all NCAM isoforms. Hybridoma clone H28 was a gift of C. Goridis (Centre National de la Recherche Scientifique UMR 8542, Paris, France). Rabbit antibodies against RPTP were a gift of C.J. Pallen (University of British Columbia, Vancouver, Canada) or were generated as described previously (den Hertog et al., 1994). Rabbit polyclonal antibodies against human erythrocyte spectrin, rabbit polyclonal antibodies against the HA tag, nonspecific rabbit immunoglobulins, and cholera toxin B subunit tagged with fluorescein to label GM1 were obtained from Sigma-Aldrich. Mouse mAbs against the HA tag (clone 12CA5) were obtained from Roche Diagnostics. Rabbit polyclonal antibodies and mouse mAbs against p59fyn protein were purchased from Santa Cruz Biotechnology, Inc. Rabbit polyclonal antibodies against Tyr-527–dephosphorylated or Tyr-416–phosphorylated pp60c-src kinase that cross-react with Tyr-531–dephosphorylated or Tyr-420–phosphorylated p59fyn were obtained from Cell Signaling Technology. Secondary antibodies against rabbit, rat, and mouse Ig coupled to HRP, Cy2, Cy3, or Cy5 were obtained from Dianova.
Animals
To compare wild-type and NCAM-deficient mice, C57BL/6J mice and NCAM-deficient mice (Cremer et al., 1994) inbred for at least nine generations onto the C57BL/6J background were used. NCAM-deficient mice were a gift of H. Cremer (Developmental Biology Institute of Marseille, Marseille, France).To compare wild-type and RPTP-deficient mice, RPTP-positive and -negative littermates obtained from heterozygous breeding were used (see online supplemental material).
Image acquisition and manipulation
Coverslips were embedded in Aqua-Poly/Mount (Polysciences, Inc.). Images were acquired at RT using a confocal laser scanning microscope (model LSM510; Carl Zeiss MicroImaging, Inc.), LSM510 software (version 3; Carl Zeiss MicroImaging, Inc.), and oil Plan-Neofluar 40x objective (NA 1.3; Carl Zeiss MicroImaging, Inc.) at 3x digital zoom. Contrast and brightness of the images were further adjusted in Photo-Paint 9 (Corel Corporation).
Detergent extraction of cultured neurons
Cells washed in PBS were incubated for 1 min in cold microtubule-stabilizing buffer (2 mM MgCl2, 10 mM EGTA, and 60 mM Pipes, pH 7.0) and extracted 8 min on ice with 1% Triton X-100 in microtubule-stabilizing buffer as described previously (Ledesma et al., 1998). After washing with PBS, cells were fixed with cold 4% formaldehyde in PBS.
Colocalization analysis
Colocalization quantification was performed as described previously (Leshchyns'ka et al., 2003). In brief, an NCAM cluster was defined as an accumulation of NCAM labeling with a mean intensity at least 30% higher than background. NCAM clusters were automatically outlined using the threshold function of the Scion Image software (Scion Corporation). Within the outlined areas the mean intensities of NCAM, RPTP, p59fyn, or GM1 labeling associated with NCAM cluster were measured. The same threshold was used for all groups. All experiments were performed two to three times with the same effect. Colocalization profiles were plotted using ImageJ software (National Institutes of Health).
DNA constructs
Rat NCAM140 and NCAM180/pcDNA3 were a gift of P. Maness (University of North Carolina, Chapel Hill, NC). Rat NCAM120 (a gift of E. Bock, University of Copenhagen, Copenhagen, Denmark) was subcloned into the pcDNA3 vector (Invitrogen) by two EcoRI sites. The EGFP plasmid was purchased from CLONTECH Laboratories, Inc. cDNAs encoding intracellular domains of NCAM140 and NCAM180 were as described previously (Sytnyk et al., 2002; Leshchyns'ka et al., 2003). The plasmid encoding the intracellular domain of RPTP was a gift of C.J. Pallen. Wild-type RPTP, RPTPC433S, and RPTPD2 (containing RPTP residues 1–516 [last 6 residues: KIYNKI]) were as described previously (den Hertog and Hunter, 1996; Blanchetot and den Hertog, 2000; Buist et al., 2000).
Online supplemental material
Details on cultures and transfection of hippocampal neurons and CHO cells, immunofluorescence labeling, ELISA and pull-down assay, coimmunoprecipitation, isolation of lipid enriched microdomains, gel electrophoresis, immunoblotting, and generation of RPTP-deficient mice are given in online supplemental material. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200405073/DC1.
Acknowledgments
We thank Achim Dahlmann and Eva Kronberg for genotyping and animal care, Drs. Patricia Maness and Elisabeth Bock for NCAM cDNAs, Dr. Catherine J. Pallen for antibodies against RPTP and cDNA encoding the RPTP intracellular domain, Dr. Harold Cremer for NCAM-deficient mice, and Dr. Christo Goridis for hybridoma clone H28.
This work was supported by Zonta Club, Hamburg-Alster (I. Leshchyns'ka), and Deutsche Forschungsgemeinschaft (I. Leshchyns'ka, V. Sytnyk, and M. Schachner).
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2 Hubrecht Laboratory, Netherlands Institute for Developmental Biology, 3584 CT Utrecht, Netherlands
The neural cell adhesion molecule (NCAM) forms a complex with p59fyn kinase and activates it via a mechanism that has remained unknown. We show that the NCAM140 isoform directly interacts with the intracellular domain of the receptor-like protein tyrosine phosphatase RPTP, a known activator of p59fyn. Whereas this direct interaction is Ca2+ independent, formation of the complex is enhanced by Ca2+-dependent spectrin cytoskeleton–mediated cross-linking of NCAM and RPTP in response to NCAM activation and is accompanied by redistribution of the complex to lipid rafts. Association between NCAM and p59fyn is lost in RPTP-deficient brains and is disrupted by dominant-negative RPTP mutants, demonstrating that RPTP is a link between NCAM and p59fyn. NCAM-mediated p59fyn activation is abolished in RPTP-deficient neurons, and disruption of the NCAM–p59fyn complex in RPTP-deficient neurons or with dominant-negative RPTP mutants blocks NCAM-dependent neurite outgrowth, implicating RPTP as a major phosphatase involved in NCAM-mediated signaling.
V. Bodrikov, I. Leshchyns'ka, and V. Sytnyk contributed equally to this paper.
Abbreviations used in this paper: FGFR, FGF receptor; NCAM, neural cell adhesion molecule.
Introduction
The neural cell adhesion molecule (NCAM) is involved in several morphogenetic events, such as neuronal migration and differentiation, neurite outgrowth, and axon fasciculation. NCAM-induced morphogenetic effects depend on activation of Src family tyrosine kinases and, in particular, p59fyn kinase (Schmid et al., 1999). NCAM-dependent neurite outgrowth is impaired in neurons from p59fyn-deficient mice (Beggs et al., 1994) and is abolished by inhibitors of Src kinase family members (Crossin and Krushel, 2000; Kolkova et al., 2000; Cavallaro et al., 2001). The NCAM140 isoform has been observed in a complex with p59fyn, whereas p59fyn does not associate significantly with NCAM180 or glycosylphosphatidylinositol-linked NCAM120 (Beggs et al., 1997). However in oligodendrocytes, p59fyn is also associated with NCAM120 in isolated lipid rafts (Kramer et al., 1999), whereas in tumor cells NCAM is also associated with pp60c-src (Cavallaro et al., 2001), suggesting that additional molecular mechanisms may define NCAM's specificity of interactions with Src kinase family members. Several lines of evidence suggest that NCAM's association with lipid rafts is critical for p59fyn activation. NCAM not only colocalizes with p59fyn in lipid rafts (He and Meiri, 2002) but disruption of NCAM140 association with lipid rafts either by mutation of NCAM140 palmitoylation sites or by lipid raft destruction attenuates activation of the p59fyn kinase pathway, completely blocking neurite outgrowth (Niethammer et al., 2002). However, in spite of compelling evidence that NCAM can activate Src family tyrosine kinases, the mechanism of this activation has remained unclear.
The activity of Src family tyrosine kinases is regulated by phosphorylation (Brown and Cooper, 1996; Thomas and Brugge, 1997; Bhandari et al., 1998; Hubbard, 1999; Petrone and Sap, 2000). The two best-characterized tyrosine phosphorylation sites in Src family tyrosine kinases perform opposing regulatory functions. The site within the enzyme's activation loop (Tyr-420 in p59fyn) undergoes autophosphorylation, which is crucial for achieving full kinase activity. In contrast, phosphorylation of the COOH-terminal site (Tyr-531 in p59fyn) inhibits kinase activity through intramolecular interaction between phosphorylated Tyr-531 and the SH2 domain in p59fyn, which stabilizes a noncatalytic conformation.
A well known activator of Src family tyrosine kinases is the receptor protein tyrosine phosphatase RPTP (Zheng et al., 1992, 2000; den Hertog et al., 1993; Su et al., 1996; Ponniah et al., 1999). It contains two cytoplasmic catalytic domains, D1 and D2, of which only D1 is significantly active in vitro and in vivo (Wang and Pallen, 1991; den Hertog et al., 1993; Wu et al., 1997; Harder et al., 1998). To activate Src family tyrosine kinase, constitutively phosphorylated pTyr789 at the COOH-terminal of RPTP binds the SH2 domain of Src family tyrosine kinase that disrupts the intra-molecular association between the SH2 and SH1 domains of the kinase. This initial binding is followed by binding between the inhibitory COOH-terminal phosphorylation site of the Src family tyrosine kinase (pTyr531 in p59fyn) and the D1 domain of RPTP resulting in dephosphorylation of the inhibitory COOH-terminal phosphorylation sites in Src family tyrosine kinases (Zheng et al., 2000). These sites are hyperphosphorylated in cells lacking RPTP, and kinase activity of pp60c-src and p59fyn in RPTP-deficient mice is reduced (Ponniah et al., 1999). Like p59fyn and NCAM, RPTP is particularly abundant in the brain (Kaplan et al., 1990; Krueger et al., 1990), accumulates in growth cones (Helmke et al., 1998), and is involved in neural cell migration and neurite outgrowth (Su et al., 1996; Yang et al., 2002; Petrone et al., 2003).
Remarkably, a close homologue of RPTP, CD45, associates with the membrane-cytoskeleton linker protein spectrin (Lokeshwar and Bourguignon, 1992; Iida et al., 1994), a binding partner of NCAM (Leshchyns'ka et al., 2003). Following this lead, we investigated the possibility that RPTP is involved in NCAM-induced p59fyn activation. We show that the intracellular domains of NCAM140 and RPTP interact directly and that this interaction is enhanced by spectrin-mediated Ca2+-dependent cross-linking of NCAM and RPTP. Levels of p59fyn associated with NCAM correlate with the ability of NCAM-associated RPTP to bind to p59fyn, and the NCAM–p59fyn complex is disrupted in RPTP-deficient brains implicating RPTP as linker molecule between NCAM and p59fyn. RPTP redistributes to lipid rafts in response to NCAM activation and RPTP levels are reduced in lipid rafts from NCAM-deficient mice, suggesting that NCAM recruits RPTP to lipid rafts to activate p59fyn. Finally, NCAM-mediated p59fyn activation is abolished in RPTP-deficient neurons and NCAM-induced neurite outgrowth is blocked in RPTP-deficient neurons or neurons transfected with dominant-negative RPTP mutants, demonstrating that RPTP is a major phosphatase involved in NCAM-mediated signaling.
Results
Activation of p59fyn is impaired in NCAM-deficient brains
Cross-linking of NCAM at the cell surface results in a rapid activation of p59fyn kinase (Beggs et al., 1997; Niethammer et al., 2002) via an unknown mechanism. To analyze whether or not NCAM deficiency may affect the activation status of p59fyn, we compared levels of activated p59fyn characterized by dephosphorylation at Tyr-531 and phosphorylation at Tyr-420 in the brains of wild-type and NCAM-deficient mice. Whereas the level of p59fyn protein was higher in brain homogenates of NCAM-deficient mice (Fig. 1 A), labeling with antibodies recognizing only p59fyn dephosphorylated at Tyr-531 or with antibodies recognizing only p59fyn phosphorylated at Tyr-420 was reduced in brain homogenates of NCAM-deficient mice (Fig. 1 B), indicating that activation of p59fyn is inhibited in NCAM-deficient brains and suggesting that NCAM is involved in the regulation of p59fyn function.
NCAM forms a complex with RPTP
The intracellular domain of NCAM does not contain sequences known to induce p59fyn activation. Thus, NCAM may form a complex with a protein, possibly a protein tyrosine phosphatase, to activate p59fyn. One possible candidate is the RPTP that dephosphorylates Tyr-531 of p59fyn (Bhandari et al., 1998) and is highly enriched in neurons and growth cones (Helmke et al.,1998). Remarkably, in RPTP-deficient cells, both dephosphorylation of the COOH-terminal tyrosine residue and autophosphorylation of the tyrosine residue within the activation loop of pp60c-src is reduced (von Wichert et al., 2003), resembling the phenotype of NCAM-deficient mice. Furthermore, a close homologue of RPTP, CD45, associates with the membrane-cytoskeleton linker protein spectrin (Lokeshwar and Bourguignon, 1992; Iida et al., 1994), a binding partner of NCAM (Leshchyns'ka et al., 2003). To investigate if NCAM interacts with RPTP, we analyzed the distribution of both proteins in cultured hippocampal neurons. NCAM and RPTP partially colocalized along neurites, and both proteins accumulated in growth cones where clusters of NCAM partially overlapped with accumulations of RPTP (Fig. 2 A). To verify whether or not NCAM interacts with RPTP, we induced clustering of NCAM at the cell surface of live hippocampal neurons by incubation with antibodies against NCAM. Clustering of NCAM enhanced overlap between NCAM and RPTP localization (mean correlation between NCAM and RPTP localization being 0.3 ± 0.05 and 0.6 ± 0.03 in neurons treated with nonspecific IgG or NCAM antibodies, respectively; Fig. 2 B), indicating that RPTP partially redistributed to NCAM clusters and suggesting that NCAM and RPTP form a complex. Because antibodies against RPTP were directed against its intracellular domain, RPTP contained in intracellular organelles could have been recognized as colocalizing with NCAM that associates with intracellular organelles of trans-Golgi network origin (Sytnyk et al., 2002). Thus, the redistribution of RPTP to NCAM clusters may represent redistribution of intracellular carriers containing RPTP. To analyze whether or not NCAM associates with RPTP in the plasma membrane, we transfected neurons with RPTP containing the HA tag in the extracellular domain and induced clustering of NCAM and HA-RPTP with antibodies against NCAM and the HA tag. HA-RPTP partially redistributed to NCAM clusters (Fig. 2 C), indicating that both proteins form a complex at the cell surface.
Finally, we examined the association between NCAM and RPTP in the brain by coimmunoprecipitation. RPTP coimmunoprecipitated with NCAM from brain homogenates (Fig. 2 D), confirming that NCAM associates with RPTP. Interestingly, we found that the level of RPTP was approximately two times higher in the brain of NCAM-deficient mice when compared with wild-type mice (Fig. 2 D), indicating a functional relationship between NCAM and RPTP.
NCAM140 is the most potent RPTP-binding NCAM isoform
To identify the NCAM isoform interacting with RPTP, we expressed NCAM120, NCAM140, and NCAM180 in CHO cells and analyzed their association with RPTP by coimmunoprecipitation. CHO cells endogenously express RPTP that was detected with RPTP antibodies as a band with a molecular mass identical to RPTP detected in brain homogenates (unpublished data). Although transfected CHO cells expressed NCAM120, NCAM140, and NCAM180 in similar amounts, RPTP coimmunoprecipitated only with NCAM140 (Fig. 3 A). However, after prolonged exposure of the film we could also detect RPTP in NCAM180 immunoprecipitates (unpublished data). RPTP did not coimmunoprecipitate with NCAM120. We conclude that RPTP associates predominantly with NCAM140 and to a lesser extent with NCAM180.
Inability of NCAM120, the GPI-linked NCAM isoform without the intracellular domain, to bind RPTP suggested that the intracellular domain of NCAM is involved in the formation of a complex between NCAM and RPTP. Furthermore, the extracellular domain of NCAM (NCAM-Fc) did not bind to RPTP in brain lysates, confirming that the extracellular domain of NCAM does not bind to RPTP (unpublished data). To verify that the NCAM intracellular domain interacts directly with the intracellular domain of RPTP, we analyzed binding of the recombinant intracellular domain of RPTP to the intracellular domain of NCAM180 or NCAM140 in a pull-down assay. For comparison, the intracellular domain of CHL1, another adhesion molecule of the immunoglobulin superfamily, was used. The intracellular domain of RPTP bound to the intracellular domain of NCAM180 or NCAM140 but not to the intracellular domain of CHL1 (Fig. 3 B). Interaction between the intracellular domains of RPTP and NCAM140 was severalfold stronger than between the intracellular domains of RPTP and NCAM180 (Fig. 3 B). To confirm this finding, we examined the direct interaction between the intracellular domains of RPTP and NCAM180 or NCAM140 by ELISA. Intracellular domain of RPTP bound to the intracellular domains of NCAM180 or NCAM140 in a concentration-dependent manner, with the intracellular domain of NCAM140 binding with a higher affinity than the intracellular domain of NCAM180 (Fig. 3 C). No binding with the intracellular domain of CHL1 was observed (Fig. 3 C). We conclude that NCAM binds directly to RPTP via the intracellular domain, with NCAM140 being the most potent RPTP-binding NCAM isoform.
RPTP binds NCAM140 via the D2 domain and links NCAM140 to p59fyn
To identify the part of the intracellular domain of RPTP responsible for the interaction with NCAM140, we coexpressed, in CHO cells, NCAM140 together with the wild-type form of RPTP (wtRPTP), RPTP lacking the D2 domain (RPTPD2), or catalytically inactive form of RPTP containing a mutation within the D1 catalytic domain (RPTPC433S) and analyzed binding of NCAM140 to these RPTP mutants by coimmunoprecipitation. All transfected RPTP constructs contained the HA tag to distinguish them from endogenous RPTP. As seen for endogenous RPTP, transfected wtRPTP coimmunoprecipitated with NCAM140 (Fig. 4 A). Similar amounts of RPTPC433S coimmunoprecipitated with NCAM140, whereas RPTPD2 did not coimmunoprecipitate (Fig. 4 A), indicating that the D2 domain is required for the interaction between RPTP and NCAM140.
Remarkably, among the major NCAM isoforms, only NCAM140 forms a complex with p59fyn (Beggs et al., 1997) that we found to correlate with its ability to bind RPTP (see the previous section). RPTP directly interacts with p59fyn (Bhandari et al., 1998). Accordingly, p59fyn coimmunoprecipitated with wtRPTP from transfected CHO cells (Fig. 4 A). Approximately the same amount of p59fyn coimmunoprecipitated with RPTPD2 (Fig. 4 A), indicating that this truncated construct also binds p59fyn probably via the D1 domain. In accordance with previous reports, p59fyn showed reduced ability to bind RPTPC433S, a catalytically inactive mutant of RPTP (Fig. 4 A; Zheng et al., 2000).
To analyze the role of RPTP in NCAM140–p59fyn complex formation, we coimmunoprecipitated p59fyn with NCAM140 in the presence of RPTP mutants. In CHO cells cotransfected with NCAM140 and wtRPTP, p59fyn coimmunoprecipitated with NCAM140 (Fig. 4 A). The amount of p59fyn coimmunoprecipitated with NCAM140 was reduced in cells cotransfected with RPTPC433S (Fig. 4 A), correlating with the reduced ability of this catalytically inactive RPTP mutant to bind p59fyn (see previous paragraph; Zheng et al., 2000). When NCAM140 was cotransfected with RPTPD2, p59fyn no longer coimmunoprecipitated with NCAM140 (Fig. 4 A). Because RPTPD2 binds p59fyn (Fig. 4 A), it is conceivable that this mutant, which does not bind NCAM140, competes with endogenous RPTP for binding to p59fyn and thus inhibits NCAM140–p59fyn complex formation.
To extend this analysis to neurons, we transfected hippocampal neurons with GFP alone or cotransfected with GFP and RPTPD2 or RPTPC433S and analyzed the redistribution of p59fyn to NCAM clusters after cross-linking NCAM with NCAM antibodies (Fig. 4 B). In neurons transfected with RPTPD2 or RPTPC433S, the level of p59fyn in NCAM clusters was reduced by 30% when compared with GFP only transfected cells, suggesting that RPTPD2 or RPTPC433S inhibit NCAM–p59fyn complex formation by competing with endogenous RPTP. The combined observations indicate that NCAM140–p59fyn complex formation correlates with the ability of NCAM140-associated RPTP to bind to p59fyn, implicating RPTP as a linker between NCAM140 and p59fyn.
Association between NCAM and p59fyn and NCAM-mediated p59fyn activation are abolished in RPTP-deficient neurons
To substantiate further our finding that RPTP is a linker protein between NCAM and p59fyn, we analyzed p59fyn activation and association of p59fyn with NCAM in RPTP-deficient brains. As for NCAM-deficient brains, levels of p59fyn dephosphorylated at Tyr-531 and levels of p59fyn phosphorylated at Tyr-420 were reduced in brain homogenates of RPTP-deficient mice (Fig. 5 A), further suggesting that RPTP plays a role in NCAM-mediated p59fyn activation in the brain. To analyze the role of RPTP in the formation of the complex between NCAM and p59fyn, we immunoprecipitated NCAM from wild-type and RPTP-deficient brains and probed immunoprecipitates with antibodies against p59fyn. Whereas p59fyn coimmunoprecipitated with NCAM from wild-type brains, p59fyn did not coimmunoprecipitate with NCAM from RPTP-deficient brains (Fig. 5 B). Furthermore, when NCAM was clustered at the surface of wild-type and RPTP-deficient cultured hippocampal neurons, levels of p59fyn were significantly reduced in NCAM clusters in RPTP-deficient neurons when compared with wild-type cells (Fig. 5 C), indicating that RPTP is required for complex formation between NCAM and p59fyn.
NCAM clustering at the cell surface induces rapid p59fyn activation (Beggs et al., 1997). To analyze whether or not RPTP is required for NCAM-induced p59fyn activation, we treated live hippocampal neurons from wild-type and RPTP-deficient mice with NCAM antibodies and analyzed levels of p59fyn dephosphorylated at Tyr-531 along neurites of the stimulated neurons. Clustering of NCAM increased levels of Tyr-531–dephosphorylated p59fyn along neurites of wild-type neurons by 60% (Fig. 5 D). However, NCAM-mediated p59fyn activation was completely abolished in RPTP-deficient neurons (Fig. 5 D), demonstrating that RPTP is required for NCAM-mediated p59fyn activation.
Formation of the complex between RPTP and NCAM is enhanced by Ca2+
Coimmunoprecipitation experiments were performed either in the presence of Ca2+ or with 2 mM EDTA, a Ca2+-sequestering agent. Whereas RPTP coimmunoprecipitated with NCAM from brain homogenates under both conditions, coimmunoprecipitated complexes were reduced by 60% in the presence of EDTA (Fig. 6 A), suggesting that Ca2+ promotes formation of the NCAM–RPTP complex. These results are in accordance with findings of Zeng et al. (1999), who found that NCAM and RPTP did not coimmunoprecipitate in the presence of EDTA. To analyze if the direct interaction between NCAM and RPTP is Ca2+ dependent, we assayed binding of the intracellular domain of NCAM140 to the intracellular domain of RPTP by ELISA in the presence or absence of Ca2+ (Fig. 6 B), showing that the direct interaction is Ca2+ independent and suggesting that additional binding partners of NCAM and/or RPTP may enhance complex formation in a Ca2+-dependent manner. Spectrin, which directly interacts with the intracellular domain of NCAM (Leshchyns'ka et al., 2003) and contains a Ca2+ binding domain (De Matteis and Morrow, 2000), is one of the possible candidates. Indeed, RPTP coimmunoprecipitated with spectrin from brain homogenates (Fig. 6 C). In the presence of 2 mM EDTA, RPTP coimmunoprecipitating with spectrin was reduced by 80% (Fig. 6 C), whereas coimmunoprecipitation of NCAM with spectrin did not depend on Ca2+ (Fig. 6 C). We conclude that RPTP directly interacts with NCAM in a Ca2+-independent manner. However, formation of the complex is enhanced by Ca2+-dependent cross-linking of NCAM140 and RPTP via spectrin.
The NCAM–RPTP complex redistributes to lipid rafts after NCAM activation
Whereas p59fyn is mainly associated with lipid rafts (van't Hof and Resh, 1997; Niethammer et al., 2002; Filipp et al., 2003), only 4–8% of all RPTP molecules were found in lipid rafts of brain (unpublished data). In hippocampal neurons extracted with cold 1% Triton X-100 to isolate lipid rafts (Niethammer et al., 2002; Leshchyns'ka et al., 2003), detergent-insoluble clusters of RPTP only partially overlapped with the lipid raft marker ganglioside GM1 (Fig. 7 A), further confirming that RPTP and p59fyn are segregated at the subcellular level. Because activation of NCAM results in its redistribution to lipid rafts (Leshchyns'ka et al., 2003), it may also promote redistribution of NCAM-associated RPTP to lipid rafts and thus activate raft-associated p59fyn. To verify this hypothesis, we studied association of NCAM and RPTP with lipid rafts in hippocampal neurons activated or not activated with NCAM-Fc or NCAM antibodies. In accordance with previous results (Leshchyns'ka et al., 2003), application of NCAM-Fc or NCAM antibodies increased GM1 levels in detergent-insoluble clusters of NCAM, indicating that NCAM redistributed to lipid rafts (Fig. 7, A–C). Application of NCAM-Fc or NCAM antibodies also increased the level of RPTP in NCAM clusters, indicating that NCAM activation promoted NCAM–RPTP complex formation (Fig. 7, A–C). Furthermore, NCAM activation also increased GM1 levels in detergent-insoluble clusters of RPTP (Fig. 7 D), confirming that NCAM-associated RPTP also redistributed to lipid rafts and suggesting that NCAM recruits RPTP to lipid rafts. To further analyze this possibility, we compared levels of RPTP in lipid rafts in brains of wild-type and NCAM-deficient mice. Indeed, RPTP was reduced by 60% in lipid rafts isolated from NCAM-deficient brains (Fig. 7 E), confirming that NCAM plays a role in RPTP targeting to lipid rafts. The levels of p59fyn were increased in NCAM-deficient lipid rafts (100% and 124 ± 7.6% in wild-type and NCAM deficient rafts, respectively) probably reflecting increased levels of p59fyn in NCAM-deficient brains. Levels of GM1 were not different in lipid rafts from wild-type and NCAM-deficient brains (100% and 103.3 ± 6.8% in wild-type and NCAM-deficient rafts, respectively), showing that lipid rafts were isolated with the same efficacy from wild-type and NCAM-deficient brains (Fig. 7 E).
NCAM-mediated recruitment of RPTP to lipid rafts is enhanced by NCAM-induced FGF receptor (FGFR)–dependent increase in intracellular Ca2+
NCAM activation increases intracellular Ca2+ concentrations via a FGFR-dependent mechanism (Walsh and Doherty, 1997; Kamiguchi and Lemmon, 2000; Juliano, 2002). This increase in intracellular Ca2+ may account for the enhanced association between NCAM and RPTP after NCAM activation (Fig. 7, A–D) because of spectrin-mediated cross-linking of NCAM140 and RPTP (Fig. 6). Interestingly, NCAM activation also induces redistribution of NCAM-associated spectrin to lipid rafts (Leshchyns'ka et al., 2003). To analyze the role of FGFR and Ca2+ in the recruitment of RPTP to an NCAM complex, we estimated levels of RPTP associated with NCAM following NCAM activation in control neurons and neurons incubated with BAPTA-AM, a membrane-permeable Ca2+ chelator (Williams et al., 1992; Cavallaro et al., 2001), or a specific FGFR inhibitor (Niethammer et al., 2002; Leshchyns'ka et al., 2003). Whereas NCAM activation increased levels of RPTP and GM1 in NCAM clusters (Fig. 7 F), treatment with BAPTA-AM or FGFR inhibitor abolished recruitment of RPTP to NCAM clusters in response to NCAM activation (Fig. 7 F). In accordance with previous findings (Leshchyns'ka et al., 2003), NCAM redistribution to lipid rafts was not affected by the FGFR inhibitor or BAPTA-AM (Fig. 7 F). BAPTA-AM or FGFR inhibitor did not affect the level of RPTP associated with NCAM under nonactivated conditions (Fig. 7 F). We conclude that, whereas at resting conditions Ca2+ does not play a major role in the interaction between NCAM and RPTP, NCAM-induced FGFR-dependent elevations of intracellular Ca2+ levels strengthen the interactions between NCAM and RPTP in response to NCAM activation, most likely via spectrin (see the section Formation of the complex between RPTP and NCAM is enhanced by Ca2+).
NCAM-induced neurite outgrowth depends on NCAM association with RPTP
NCAM-induced neurite outgrowth depends on p59fyn activation (Kolkova et al., 2000), suggesting that NCAM association with RPTP may be involved. To analyze the role of protein tyrosine phosphatases in NCAM-induced neurite outgrowth, we incubated cultured hippocampal neurons with 100 μM vanadate, an inhibitor of these phosphatases (Helmke et al., 1998). NCAM-Fc–enhanced neurite outgrowth was abolished by vanadate, indicating that activation of protein tyrosine phosphatases is required for NCAM-mediated neurite outgrowth. Vanadate did not affect neurite outgrowth in nonstimulated neurons, indicating that vanadate does not lead to nonspecific impairments (Fig. 8 A). To directly assess the role of RPTP in NCAM-induced neurite outgrowth, we transfected hippocampal neurons with the dominant-negative mutants of RPTP. Both, RPTPD2, which does not bind NCAM but associates with p59fyn, and catalytically inactive RPTPC433S, which associates with NCAM but binds p59fyn with a lower efficiency than endogenous RPTP, inhibited association of NCAM with p59fyn by competing with endogenous RPTP (Fig. 4). In neurons transfected with GFP only, stimulation with NCAM-Fc significantly enhanced neurite length when compared with control nonstimulated neurons (Fig. 8 B). However, neurons transfected with RPTPD2 or RPTPC433S remained unresponsive to NCAM-Fc stimulation (Fig. 8 B), indicating that RPTP plays a major role in NCAM-induced neurite outgrowth. To confirm this finding, we analyzed NCAM-mediated neurite outgrowth in hippocampal neurons from RPTP-deficient mice. Whereas NCAM-Fc enhanced neurite outgrowth in neurons from RPTP wild-type littermates by 100%, NCAM-Fc–induced neurite outgrowth was completely abolished in RPTP-deficient neurons (Fig. 8 C), further confirming that RPTP is required for NCAM-mediated neurite outgrowth.
Discussion
It is by now well established that in response to homophilic or heterophilic binding cell adhesion molecules of the immunoglobulin superfamily, such as NCAM, L1, or CHL1, activate Src family tyrosine kinases, and in particular pp60c-src or p59fyn, resulting in morphogenetic events, such as cell migration and neurite outgrowth. However, the mechanisms of Src family tyrosine kinase activation in these paradigms have remained unresolved. Here, we identify a cognate activator of p59fyn, the receptor protein tyrosine phosphatase RPTP, as a novel binding partner of NCAM. Activation of p59fyn is reduced in NCAM-deficient mice and interaction between NCAM and p59fyn is abolished in RPTP-deficient brains. Interestingly, we found that the levels of p59fyn and RPTP are increased in NCAM-deficient brains, possibly reflecting a compensatory reaction to the decreased activity of these enzymes in the mutant and further indicating a tight functional relationship between NCAM, RPTP, and p59fyn. NCAM-induced neurite outgrowth is completely abrogated in RPTP-deficient neurons or in neurons transfected with dominant-negative RPTP mutants, indicating that RPTP links NCAM to p59fyn both physically and functionally.
Role of Ca2+ in NCAM–RPTP–p59fyn complex formation
Interactions between NCAM and RPTP and NCAM–RPTP–p59fyn complex formation leading to neurite outgrowth are tightly regulated (Fig. 9). First, whereas direct interaction between NCAM and RPTP is Ca2+ independent, NCAM–RPTP complex formation is enhanced by Ca2+-dependent cross-linking via spectrin. Remarkably, NCAM activation results in an increase in intracellular Ca2+ concentration via influx through Ca2+ channels or release from intracellular stores, and may thus provide a positive feedback loop between NCAM activation and NCAM–RPTP complex formation involving spectrin. RPTP binding to spectrin may also elevate RPTP enzymatic dephosphorylation activity (Lokeshwar and Bourguignon, 1992). Interestingly, NCAM activation also induces activation of PKC (Kolkova et al., 2000; Leshchyns'ka et al., 2003), which is known to phosphorylate RPTP and stimulate its activity (den Hertog et al., 1995; Tracy et al., 1995; Zheng et al., 2002). Thus, a network of activated intracellular signaling molecules may underlie the induction and maintenance of NCAM-mediated neurite outgrowth. It is interesting in this respect that the NCAM140 isoform predominates in these interactions: it interacts more efficiently with p59fyn via RPTP and enhances neurite outgrowth more vigorously than NCAM180 (Niethammer et al., 2002). The structural dispositions of NCAM140 for this preference will remain to be established.
The role of lipid rafts
Additional regulation of RPTP-mediated p59fyn activation is achieved by segregation of RPTP and p59fyn to different subdomains in the plasma membrane (Fig. 9). Approximately 90% of all RPTP molecules in the brain are located in a lipid raft-free environment and are thereby segregated from lipid raft-associated p59fyn under nonstimulated conditions. Whereas segregation of receptor protein tyrosine phosphatases from their potential substrates due to targeting to different plasma membrane domains has been suggested as a general mechanism of the regulation of receptor protein tyrosine phosphatase function (Petrone and Sap, 2000), the mechanisms that target receptor protein tyrosine phosphatases to lipid rafts have remained unclear. We show that levels of raft-associated RPTP in the NCAM-deficient brain are reduced, and NCAM redistribution to lipid rafts in response to NCAM activation also induces redistribution of RPTP to lipid rafts via its NCAM association. The combined observations indicate that NCAM plays a role in recruiting NCAM-associated RPTP to lipid rafts via NCAM palmitoylation (Niethammer et al., 2002; Fig. 9) or via NCAM interaction with GPI-anchored components of lipid rafts, such as the GPI-linked GDNF receptor (Paratcha et al., 2003). Investigations on the regulatory mechanisms underlying palmitoylation will be required to understand the subcellular compartment-specific distribution of the NCAM–RPTP–p59fyn complex. Furthermore, the localization of adhesion molecules and receptors will have to be elucidated in view of lipid rafts heterogeneities.
Potential role of protein tyrosine phosphatases in signaling mediated by other cell adhesion molecules
Besides NCAM, activation of L1 and CHL1, other cell adhesion molecules of the immunoglobulin superfamily, also results in the activation of Src family tyrosine kinases (Schmid et al., 2000; Buhusi et al., 2003). As for NCAM, the intracellular domains of L1 and CHL1 do not possess structural motif for protein tyrosine phosphatase activity, suggesting that yet unidentified protein tyrosine phosphatases may be involved. Identification of protein tyrosine phosphatases associated with other cell adhesion molecules of the immunoglobulin superfamily and conjunctions with RPTP-activated integrins (Zeng et al., 2003) will be an important next step in the elucidation of the mechanisms that cell adhesion molecules use differentially to guide cell migration and neurite outgrowth in the developing nervous system.
Materials and methods
Antibodies and toxins
Rabbit polyclonal antibodies against NCAM (Niethammer et al., 2002) were used in immunoprecipitation, immunoblotting, and immunocytochemical experiments; and rat mAbs H28 against mouse NCAM (Gennarini et al., 1984) were used in immunocytochemical experiments. Both antibodies recognize the extracellular domain of all NCAM isoforms. Hybridoma clone H28 was a gift of C. Goridis (Centre National de la Recherche Scientifique UMR 8542, Paris, France). Rabbit antibodies against RPTP were a gift of C.J. Pallen (University of British Columbia, Vancouver, Canada) or were generated as described previously (den Hertog et al., 1994). Rabbit polyclonal antibodies against human erythrocyte spectrin, rabbit polyclonal antibodies against the HA tag, nonspecific rabbit immunoglobulins, and cholera toxin B subunit tagged with fluorescein to label GM1 were obtained from Sigma-Aldrich. Mouse mAbs against the HA tag (clone 12CA5) were obtained from Roche Diagnostics. Rabbit polyclonal antibodies and mouse mAbs against p59fyn protein were purchased from Santa Cruz Biotechnology, Inc. Rabbit polyclonal antibodies against Tyr-527–dephosphorylated or Tyr-416–phosphorylated pp60c-src kinase that cross-react with Tyr-531–dephosphorylated or Tyr-420–phosphorylated p59fyn were obtained from Cell Signaling Technology. Secondary antibodies against rabbit, rat, and mouse Ig coupled to HRP, Cy2, Cy3, or Cy5 were obtained from Dianova.
Animals
To compare wild-type and NCAM-deficient mice, C57BL/6J mice and NCAM-deficient mice (Cremer et al., 1994) inbred for at least nine generations onto the C57BL/6J background were used. NCAM-deficient mice were a gift of H. Cremer (Developmental Biology Institute of Marseille, Marseille, France).To compare wild-type and RPTP-deficient mice, RPTP-positive and -negative littermates obtained from heterozygous breeding were used (see online supplemental material).
Image acquisition and manipulation
Coverslips were embedded in Aqua-Poly/Mount (Polysciences, Inc.). Images were acquired at RT using a confocal laser scanning microscope (model LSM510; Carl Zeiss MicroImaging, Inc.), LSM510 software (version 3; Carl Zeiss MicroImaging, Inc.), and oil Plan-Neofluar 40x objective (NA 1.3; Carl Zeiss MicroImaging, Inc.) at 3x digital zoom. Contrast and brightness of the images were further adjusted in Photo-Paint 9 (Corel Corporation).
Detergent extraction of cultured neurons
Cells washed in PBS were incubated for 1 min in cold microtubule-stabilizing buffer (2 mM MgCl2, 10 mM EGTA, and 60 mM Pipes, pH 7.0) and extracted 8 min on ice with 1% Triton X-100 in microtubule-stabilizing buffer as described previously (Ledesma et al., 1998). After washing with PBS, cells were fixed with cold 4% formaldehyde in PBS.
Colocalization analysis
Colocalization quantification was performed as described previously (Leshchyns'ka et al., 2003). In brief, an NCAM cluster was defined as an accumulation of NCAM labeling with a mean intensity at least 30% higher than background. NCAM clusters were automatically outlined using the threshold function of the Scion Image software (Scion Corporation). Within the outlined areas the mean intensities of NCAM, RPTP, p59fyn, or GM1 labeling associated with NCAM cluster were measured. The same threshold was used for all groups. All experiments were performed two to three times with the same effect. Colocalization profiles were plotted using ImageJ software (National Institutes of Health).
DNA constructs
Rat NCAM140 and NCAM180/pcDNA3 were a gift of P. Maness (University of North Carolina, Chapel Hill, NC). Rat NCAM120 (a gift of E. Bock, University of Copenhagen, Copenhagen, Denmark) was subcloned into the pcDNA3 vector (Invitrogen) by two EcoRI sites. The EGFP plasmid was purchased from CLONTECH Laboratories, Inc. cDNAs encoding intracellular domains of NCAM140 and NCAM180 were as described previously (Sytnyk et al., 2002; Leshchyns'ka et al., 2003). The plasmid encoding the intracellular domain of RPTP was a gift of C.J. Pallen. Wild-type RPTP, RPTPC433S, and RPTPD2 (containing RPTP residues 1–516 [last 6 residues: KIYNKI]) were as described previously (den Hertog and Hunter, 1996; Blanchetot and den Hertog, 2000; Buist et al., 2000).
Online supplemental material
Details on cultures and transfection of hippocampal neurons and CHO cells, immunofluorescence labeling, ELISA and pull-down assay, coimmunoprecipitation, isolation of lipid enriched microdomains, gel electrophoresis, immunoblotting, and generation of RPTP-deficient mice are given in online supplemental material. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200405073/DC1.
Acknowledgments
We thank Achim Dahlmann and Eva Kronberg for genotyping and animal care, Drs. Patricia Maness and Elisabeth Bock for NCAM cDNAs, Dr. Catherine J. Pallen for antibodies against RPTP and cDNA encoding the RPTP intracellular domain, Dr. Harold Cremer for NCAM-deficient mice, and Dr. Christo Goridis for hybridoma clone H28.
This work was supported by Zonta Club, Hamburg-Alster (I. Leshchyns'ka), and Deutsche Forschungsgemeinschaft (I. Leshchyns'ka, V. Sytnyk, and M. Schachner).
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