Phosphorylation of Microtubule-associated Protein STOP by Calmodulin Kinase II

STOP proteins are microtubule-associated, calmodulinregulated proteins responsible for the high degree of stabilization displayed by neuronal microtubules. STOP suppression in mice induces synaptic defects affecting both short and long term synaptic plasticity in hippocampal neurons. Interestingly, STOP has been identified as a component of synaptic structures in neurons, despite the absence of microtubules in nerve terminals, indicating the existence of mechanisms able to induce a translocation of STOP from microtubules to synaptic compartments. Here we have tested STOP phosphorylation as a candidate mechanism for STOP relocalization. We show that, both in vitro and in vivo, STOP is phosphorylated by the multifunctional enzyme calcium/calmodulin-dependent protein kinase II (CaMKII), which is a key enzyme for synaptic plasticity. This phosphorylation occurs on at least two independent sites. Phosphorylated forms of STOP do not bind microtubules in vitro and do not co-localize with microtubules in cultured differentiating neurons. Instead, phosphorylated STOP co-localizes with actin assemblies along neurites or at branching points. Correlatively, we find that STOP binds to actin in vitro. Finally, in differentiated neurons, phosphorylated STOP co-localizes with clusters of synaptic proteins, whereas unphosphorylated STOP does not. Thus, STOP phosphorylation by CaMKII may promote STOP translocation from microtubules to synaptic compartments where it may interact with actin, which could be important for STOP function in synaptic plasticity. STOP proteins are microtubule-associated, calmodulinregulated proteins responsible for the high degree of stabilization displayed by neuronal microtubules. STOP suppression in mice induces synaptic defects affecting both short and long term synaptic plasticity in hippocampal neurons. Interestingly, STOP has been identified as a component of synaptic structures in neurons, despite the absence of microtubules in nerve terminals, indicating the existence of mechanisms able to induce a translocation of STOP from microtubules to synaptic compartments. Here we have tested STOP phosphorylation as a candidate mechanism for STOP relocalization. We show that, both in vitro and in vivo, STOP is phosphorylated by the multifunctional enzyme calcium/calmodulin-dependent protein kinase II (CaMKII), which is a key enzyme for synaptic plasticity. This phosphorylation occurs on at least two independent sites. Phosphorylated forms of STOP do not bind microtubules in vitro and do not co-localize with microtubules in cultured differentiating neurons. Instead, phosphorylated STOP co-localizes with actin assemblies along neurites or at branching points. Correlatively, we find that STOP binds to actin in vitro. Finally, in differentiated neurons, phosphorylated STOP co-localizes with clusters of synaptic proteins, whereas unphosphorylated STOP does not. Thus, STOP phosphorylation by CaMKII may promote STOP translocation from microtubules to synaptic compartments where it may interact with actin, which could be important for STOP function in synaptic plasticity. Microtubules are vital components of the cell cytoskeleton, being involved in cell mitosis, intracellular organization, and cell morphogenesis. Neurons contain abundant subpopulations of stable microtubules that resist depolymerizing conditions such as exposure to cold temperature and to depolymerizing drugs (1Baas P.W. Slaughter T. Brown A. Black M.M. J. Neurosci. Res. 1991; 30: 134-153Crossref PubMed Scopus (113) Google Scholar, 2Guillaud L. Bosc C. Fourest-Lieuvin A. Denarier E. Pirollet F. Lafanechère L. Job D. J. Cell Biol. 1998; 142: 167-179Crossref PubMed Scopus (107) Google Scholar, 3Job D. Margolis R.L. Biochemistry. 1984; 23: 3025-3031Crossref PubMed Scopus (15) Google Scholar). In neurons, microtubule stabilization is mainly because of association with a family of proteins known as STOPs 2The abbreviations used are: STOP, stable tubule only polypeptide; CE, calcium eluate; E-STOP, Early STOP; N-STOP, neuronal adult STOP; STOP-P, phosphorylated STOP; PSD, post-synaptic density; Mes, 4-morpholineethanesulfonic acid; Pipes, 1,4-piperazinediethanesulfonic acid. (stable tubule only polypeptides). STOP proteins were initially characterized as microtubule cold-stabilizing factors whose activity was inhibited by interaction with Ca2+-calmodulin (4Job D. Fischer E.H. Margolis R.L. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 4679-4682Crossref PubMed Scopus (108) Google Scholar), and subsequent work has shown that STOPs contain bifunctional modules comprised of overlapping calmodulin-binding and microtubule-stabilizing sequences (5Bosc C. Frank R. Denarier E. Ronjat M. Schweitzer A. Wehland J. Job D. J. Biol. Chem. 2001; 276: 30904-30913Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Neurons contain two major variants of STOP, E-STOP (89 kDa) and N-STOP (116 kDa). E-STOP is present in mouse brain from embryonic stage E16 to adulthood, whereas N-STOP appears at birth and is subsequently expressed in the adult brain (2Guillaud L. Bosc C. Fourest-Lieuvin A. Denarier E. Pirollet F. Lafanechère L. Job D. J. Cell Biol. 1998; 142: 167-179Crossref PubMed Scopus (107) Google Scholar, 6Bosc C. Cronk J.D. Pirollet F. Watterson D.M. Haiech J. Job D. Margolis R.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2125-2130Crossref PubMed Scopus (95) Google Scholar). STOP function has been investigated in the whole animal by studying STOP null mice (7Andrieux A. Salin P.A. Vernet M. Kujala P. Baratier J. Gory-Fauré S. Bosc C. Pointu H. Proietto D. Schweitzer A. Denarier E. Klumperman J. Job D. Genes Dev. 2002; 16: 2350-2364Crossref PubMed Scopus (141) Google Scholar). In these mice, microtubule cold stability is suppressed, with no dramatic consequences for mouse organogenesis, viability or brain anatomy. However, STOP-/- mice display multiple synaptic deficits that affect both long and short term synaptic plasticity in the hippocampus. These synaptic defects are associated with depleted vesicular pools in glutamatergic nerve terminals and with severe behavioral disorders (7Andrieux A. Salin P.A. Vernet M. Kujala P. Baratier J. Gory-Fauré S. Bosc C. Pointu H. Proietto D. Schweitzer A. Denarier E. Klumperman J. Job D. Genes Dev. 2002; 16: 2350-2364Crossref PubMed Scopus (141) Google Scholar), which, interestingly, are specifically alleviated with long term neuroleptic treatment. It has been very surprising that a protein associated with microtubules along whole neurites turns out to be important for glutamatergic synaptic function, despite apparent microtubule absence in nerve terminals. However, from proteomic analysis of synaptosomal fractions, there is evidence that STOP localizes to synapses (7Andrieux A. Salin P.A. Vernet M. Kujala P. Baratier J. Gory-Fauré S. Bosc C. Pointu H. Proietto D. Schweitzer A. Denarier E. Klumperman J. Job D. Genes Dev. 2002; 16: 2350-2364Crossref PubMed Scopus (141) Google Scholar, 9Peng J. Kim M.J. Cheng D. Duong D.M. Gygi S.P. Sheng M. J. Biol. Chem. 2004; 279: 21003-21011Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar), and this raises questions concerning the mechanisms that could promote STOP dissociation from microtubules and relocalization in synaptic structures. Previous evidence has indicated that STOP can be inactivated by calmodulin-dependent phosphorylation (10Job D. Rauch C.T. Fischer E.H. Margolis R.L. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 3894-3898Crossref PubMed Scopus (36) Google Scholar), correlated with the presence of putative CaMKII phosphorylation sites in the STOP sequence (6Bosc C. Cronk J.D. Pirollet F. Watterson D.M. Haiech J. Job D. Margolis R.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2125-2130Crossref PubMed Scopus (95) Google Scholar). Interestingly, long term potentiation, which is known to involve CaMKII, is severely impaired in STOP null mice (7Andrieux A. Salin P.A. Vernet M. Kujala P. Baratier J. Gory-Fauré S. Bosc C. Pointu H. Proietto D. Schweitzer A. Denarier E. Klumperman J. Job D. Genes Dev. 2002; 16: 2350-2364Crossref PubMed Scopus (141) Google Scholar). Here, we have tested both the occurrence and the consequences of STOP phosphorylation. We show that CaMKII phosphorylates STOP on at least two independent sites (Ser-198, Ser-491), both in vitro and in vivo. Consequently, phosphorylated STOPs do not bind to microtubules in vitro. Additionally, the phosphorylated forms of STOP co-localize with actin-rich structures in cultured neurons and bind to polymerized actin in vitro. Finally, phosphorylated STOP protein associates with synaptic markers following synaptogenesis, in cultured neurons. Thus, phosphorylation by CaMKII may delocalize STOPs from microtubules and allow STOP association with synaptic actin, which may be important for synaptic plasticity. Purification of STOP and CaMKII from Mouse Brain (Calcium Eluate (CE) Fraction)—Purification of STOP protein from adult mice brain was performed as described by Pirollet et al. (11Pirollet F. Rauch C.T. Job D. Margolis R.L. Biochemistry. 1989; 28: 835-842Crossref PubMed Scopus (36) Google Scholar). All buffers used during STOP purification contained 2 mm dithiothreitol and protease inhibitors (1 mm phenylmethylsulfonyl fluoride, 104 units/ml aprotinin; 20 μm leupeptin). Briefly, 40 brains from OF 1 mice were homogenized in MEM buffer (100 mm Mes, 1 mm MgCl2,1mm EGTA, pH 6.75) and centrifuged at 100,000 × g for 40 min at 4 °C. The supernatant was loaded on a Sepharose-S Fast Flow column and bound proteins were eluted in MEM containing 0.4 m NaCl. The eluate was adjusted to 2 mm calcium and passed through a calmodulinagarose column equilibrated in MEM buffer containing 2 mm CaCl2, 0.4 m NaCl. Bound proteins were eluted in MEM buffer containing 0.4 m NaCl and 1 mm EGTA. The purified fraction (CE) was stored in elution buffer containing 10% glycerol, at -80 °C. Phosphorylation Reaction—CE fraction containing STOPs was buffer-exchanged to 50 mm Pipes, 10 mm MgCl2, pH 7.0. The phosphorylation reaction was performed in the presence of 100 μm ATP (added of 2 μCi [γ-32P]ATP when indicated), 0.7 mm CaCl2, 0.3 μm calmodulin, 0.4 mm EGTA, 0.5 μm mycrocystine, 1 μm okadaic acid, and 5 mm 2-mercaptoethanol, during 30 min at 30 °C. For quantitative experiments, gels were dried before exposure on a screen for analysis with a PhosphorImager (Molecular Imager® FX, Bio-Rad). Relative amounts of incorporated radioactivity were quantified using Quantity One® software (Bio-Rad). For control experiments, the buffer-exchanged CE fraction was incubated with alkaline phosphatase for 30 min at 37 °C, resulting in dephosphorylation of STOPs. For experiments using preactivated CaMKII, CE fraction was incubated with 30 μm calmodulin and 0.7 mm calcium for 10 min at 30 °C. Phosphorylation reaction was then performed in the presence or in the absence of 2.5 mm EGTA at 30 °C for 30 min. Phospho-STOP Antibodies and Primary Antibodies—Rabbit polyclonal antibodies were raised against phosphosynthetic peptides corresponding to the potential phosphorylated sites in N-STOP protein, as shown in Fig. 1A. The phosphopeptides P1-P4 used for the immunization were the following: RPRpSEYQPSDAPFC (amino acids 136-148), RPQpSQERGPMQLC (amino acids 195-206), RAWpTDIKPVKPIKC (amino acids 435-447), RRIRpSLYSEPFKEC (amino acids 487-499), according to amino acid numbering of mouse N-STOP (GenBank accession number CAA75930). These peptides were used to generate sera P1, P2, P3, and P4, respectively (Eurogentec). The C-terminal cysteine residue of each peptide was introduced for the purification steps. Crude sera were affinity-purified against the phosphorylated peptide on Sulfolink coupling gel (Pierce). Antibodies were eluted with 100 mm glycine, pH 2.5, neutralized with 1 m Tris, pH 8.0, and then incubated for 1 h with the corresponding non-phosphorylated peptide coupled on Sulfolink gel to absorb antibodies reacting with non-phosphorylated peptides. Polyclonal antibodies reacting only with phosphorylated peptides were collected in the flow-through and stored at -80 °C in 50% glycerol. Affinity-purified P2 and P4 polyclonal antibodies did not show detectable cross-reactivity in enzyme-linked immunosorbent assay tests (not shown). The same phosphopeptides P1-P4 were used to generate monoclonal antibodies following standard procedure. Hybridoma culture supernatants were screened for anti-STOP phosphopeptides using enzyme-linked immunosorbent assay. The positive were and screened on for on phosphorylated and non-phosphorylated STOP were by The following antibodies were used in this against C. L. A. C. Vernet M. Schweitzer A. Pointu H. Bosc C. Salin P.A. Job D. Wehland J. Proc. Natl. Acad. Sci. U. S. A. PubMed Google Scholar), for against C-terminal of N-STOP (6Bosc C. Cronk J.D. Pirollet F. Watterson D.M. Haiech J. Job D. Margolis R.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2125-2130Crossref PubMed Scopus (95) Google 20 for and 1 for affinity-purified P2 and P4 antibodies 20 for and 1 for against culture for against CaMKII and were used following of STOPs with Microtubules or proteins were at × g for min in a at 4 °C STOP proteins of the CE fraction were phosphorylated by CaMKII or with alkaline phosphatase as described assay was performed as in and D. J. Cell Biol. PubMed Scopus Google using microtubules as assay was performed with CE fraction containing 0.5 μm or on using known amounts of STOP as or not with of and in 100 buffer 50 mm 5 mm 5 mm 1 m 50 mm MgCl2, pH in the presence of 1 mm EGTA. min of at °C, were centrifuged at × g for min at 4 °C. were and the were with buffer and in 100 of buffer was to the and to the amounts of each were by and with or on and with or CaMKII Cell cell were according to et al. J. Neurosci. PubMed Google Scholar). Briefly, mouse were and in in at 37 °C for min. were at a of on in the was to containing and of CaMKII, was to neuronal culture for h to cell when In experiments, neuronal were to for 10 In experiments, neurons in culture were by exposure to 100 10 for min to on for were 20 min with of m in pH and then for min using in pH were for by with antibodies for 1 h in 20 and then with antibodies or from and for 40 min. (Molecular was with the The were with were using a and software CaMKII Phosphorylation in amino acids sequence for CaMKII phosphorylation is where or are phosphorylated P. J. Biol. Chem. Full Text PDF PubMed Google Scholar). for such sequences in N-STOP protein sequence (GenBank number candidate sequences Fig. containing or and N-STOP two of microtubule-stabilizing and and multiple calmodulin-binding sites (5Bosc C. Frank R. Denarier E. Ronjat M. Schweitzer A. Wehland J. Job D. J. Biol. Chem. 2001; 276: 30904-30913Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The of P1-P4 sites to these STOP (5Bosc C. Frank R. Denarier E. Ronjat M. Schweitzer A. Wehland J. Job D. J. Biol. Chem. 2001; 276: 30904-30913Abstract Full Text Full Text PDF PubMed Scopus (35) Google are shown in Fig. 1A. All P1-P4 sequences are or are overlapping a calmodulin-binding on and are or are overlapping with microtubule-stabilizing modules and STOP Phosphorylation by CaMKII in used to assay STOP protein phosphorylation in vitro. These protein were purified from mouse brain by by calmodulin The in the of calmodulin-binding proteins as shown on gels in Fig. CE contained major at and at to of CE with and with the to N-STOP, and the and to of Thus, CE contained both N-STOP and CE were with and antibodies with (not shown). of CE fraction proteins with [γ-32P]ATP in the absence of Ca2+-calmodulin by analysis and in the corresponding to N-STOP or to CaMKII was in the presence of Ca2+-calmodulin with apparent of phosphorylated which at corresponding to apparent of both N-STOP and CaMKII following phosphorylation were on The whole N-STOP was that STOP been phosphorylated during the phosphorylation In a analysis of STOP phosphorylation and STOP phosphorylation a 10 min. 1 min of a of N-STOP was detectable that STOP incorporated at least of at this to quantitative the of in N-STOP at the at 1 min was that the to the of a in STOP this may the phosphorylation of the CaMKII phosphorylation sites at the We used purified N-STOP and CaMKII to N-STOP was a for We both N-STOP phosphorylation and following STOP phosphorylation with CaMKII Fig. However, STOP is to in amounts and be subsequent experiments were using CE of STOP Phosphorylation by of P1-P4 putative STOP phosphorylation sites with calmodulin-binding sites on N-STOP raised the of of STOP phosphorylation through in the presence of of Ca2+-calmodulin We STOP phosphorylation in CE in the presence of amounts of calmodulin from to 30 Full of CaMKII for calmodulin μm as indicated by the of CaMKII STOP phosphorylation was μm calmodulin as indicated by the of N-STOP on However, at calmodulin 0.3 there was a in N-STOP indicating of N-STOP phosphorylation by calmodulin in the CaMKII is preactivated through in the presence of the enzyme following as long as dephosphorylation does not We of this CaMKII for the of N-STOP phosphorylation by Ca2+-calmodulin CaMKII was to CE fractions, in the presence of 30 μm and in the presence or in the absence of EGTA to or not the Ca2+-calmodulin The N-STOP was in the absence of EGTA Ca2+-calmodulin with the in the presence of EGTA Ca2+-calmodulin these show that STOP phosphorylation is inhibited by Ca2+-calmodulin to N-STOP, with of P1-P4 with calmodulin-binding on the N-STOP Phosphorylated N-STOP to Microtubules in investigated the phosphorylation of STOP by CaMKII could affect STOP to as phosphorylation the on microtubules of associated proteins S. Biol. PubMed Scopus Google Scholar, C. J. J. PubMed Scopus Google Scholar). CE fractions, with or were with microtubules. Microtubules were then and the STOP of both supernatant and was on In the absence of STOP with being present in microtubule and in supernatants 1 and In phosphorylated STOP was in microtubule being only present in the supernatants 5 and Thus, phosphorylated STOP does not bind to microtubules in vitro indicating a by CaMKII of the STOP to microtubule in vivo. Phosphorylated STOP phosphopeptides corresponding to the potential CaMKII phosphorylation sequences on STOP Fig. and used to polyclonal or monoclonal antibodies to phosphorylated forms of of CE fractions, P2 and P4 polyclonal antibodies with N-STOP The was when a phosphorylation was introduced to and was when CE were to alkaline phosphatase we a monoclonal P1, which only very with N-STOP in CE fractions, whereas a was when a phosphorylation was introduced to These that at least of the P1-P4 peptides are phosphorylated by CaMKII in at and Additionally, the of P2 and P4 antibodies on CE the presence of phosphorylated STOP protein in adult brain This that STOP is phosphorylated at least on and in vivo. Phosphorylated STOP with in used of the polyclonal P2 and N-STOP monoclonal to phosphorylated STOP and N-STOP in neuronal The of P2 for STOP in neuronal culture was by and neurons Fig. neurons from were cultured for in a stage of at which N-STOP is this stage of neurons are a and branching J. Neurosci. 2001; PubMed Google where microtubules are N-STOP of STOP and in neurites whereas phosphorylated STOP using P2 a not with and in structures to neurites branching N-STOP with microtubules In P2 was in a the cell not with microtubules neuronal actin in along neurites and in at branching J. Neurosci. 2001; PubMed Google Scholar), we investigated the of a of phosphorylated STOP with P2 with actin in branching P2 the of the actin-rich Interestingly, which in a of the actin a of P2 indicating association of phosphorylated STOP with assemblies neurons were by a exposure to to the P2 of with CaMKII and STOP phosphorylation CaMKII phosphorylation sites can be for the protein which is present in synapses and is involved in synaptic plasticity J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar). kinase is to the In neuronal exposure to the P2 indicating of STOP phosphorylation This against a of protein kinase in STOP in vivo. We then tested STOP could interact with actin in using standard CE STOP fractions, phosphorylated or with alkaline were with actin polymerized actin was and STOP was on in both supernatants and The of at which of STOP with actin were of and μm for unphosphorylated and phosphorylated STOP, respectively and In the same CaMKII with when whereas phosphorylated CaMKII in the as from T. PubMed Scopus Google Scholar). Affinity-purified STOP fractions, of detectable amounts of CaMKII bound to indicating that actin is a of These that STOP and phosphorylated STOP both interact with polymerized actin in with Phosphorylated of STOP with of in neurons were cultured for in a stage of at which neurons are a and are by In such synaptic proteins corresponding to synaptic proteins are both present in and at In differentiated neurons, the protein and the post-synaptic protein a these phosphorylated STOP was in clusters and phosphorylated STOP with or This was with these are with a presence of phosphorylated STOP in both the and post-synaptic compartments. STOP proteins are important for synaptic as by the synaptic defects in both long and short term plasticity by mice (7Andrieux A. Salin P.A. Vernet M. Kujala P. Baratier J. Gory-Fauré S. Bosc C. Pointu H. Proietto D. Schweitzer A. Denarier E. Klumperman J. Job D. Genes Dev. 2002; 16: 2350-2364Crossref PubMed Scopus (141) Google Scholar). the apparent absence of microtubules in nerve terminals, it has been surprising to find a synaptic function for a protein associated with microtubules in neurites (2Guillaud L. Bosc C. Fourest-Lieuvin A. Denarier E. Pirollet F. Lafanechère L. Job D. J. Cell Biol. 1998; 142: 167-179Crossref PubMed Scopus (107) Google Scholar). STOP function in synaptic plasticity may from of microtubule and resulting on synaptic (7Andrieux A. Salin P.A. Vernet M. Kujala P. Baratier J. Gory-Fauré S. Bosc C. Pointu H. Proietto D. Schweitzer A. Denarier E. Klumperman J. Job D. Genes Dev. 2002; 16: 2350-2364Crossref PubMed Scopus (141) Google Scholar). However, there are for a synaptic of which are present in synaptosomal (7Andrieux A. Salin P.A. Vernet M. Kujala P. Baratier J. Gory-Fauré S. Bosc C. Pointu H. Proietto D. Schweitzer A. Denarier E. Klumperman J. Job D. Genes Dev. 2002; 16: 2350-2364Crossref PubMed Scopus (141) Google and in the J. Kim M.J. Cheng D. Duong D.M. Gygi S.P. Sheng M. J. Biol. Chem. 2004; 279: 21003-21011Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). Here, we find that CaMKII, which is a key enzyme for synaptic plasticity J. H. H. Neurosci. 2002; PubMed Scopus Google Scholar, S. J. Neurosci. PubMed Scopus Google Scholar), can STOP to microtubules through a mechanism for STOP dissociation from microtubules and in during synaptic N-STOP is phosphorylated by CaMKII in vitro on at least independent sites and of CaMKII phosphorylation sites in the Previous have shown that N-STOP interaction with microtubules can be inhibited by Ca2+-calmodulin to microtubules (4Job D. Fischer E.H. Margolis R.L. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 4679-4682Crossref PubMed Scopus (108) Google Scholar). These a in which N-STOP from microtubules in two by Ca2+-calmodulin in to by synaptic or through phosphorylation by CaMKII, in the absence of The mechanism could be during short or long term synaptic which both involve CaMKII, the to In cultured neurons, we find that phosphorylated STOP co-localizes with actin along neurites and at branching during We find that both phosphorylated and non-phosphorylated forms of STOP bind to polymerized actin in phosphorylated STOP has for In the of is μm L. PubMed Scopus Google Scholar), which is the to absorb both and phosphorylated STOP in vitro and It is STOP to actin at actin or at branching is important because we have not of in STOP null mice (7Andrieux A. Salin P.A. Vernet M. Kujala P. Baratier J. Gory-Fauré S. Bosc C. Pointu H. Proietto D. Schweitzer A. Denarier E. Klumperman J. Job D. Genes Dev. 2002; 16: 2350-2364Crossref PubMed Scopus (141) Google Scholar). major of STOP is a dramatic of synaptic pools in glutamatergic which could be to of the synaptic disorders in STOP null mice (7Andrieux A. Salin P.A. Vernet M. Kujala P. Baratier J. Gory-Fauré S. Bosc C. Pointu H. Proietto D. Schweitzer A. Denarier E. Klumperman J. Job D. Genes Dev. 2002; 16: 2350-2364Crossref PubMed Scopus (141) Google Scholar). is important for the and of synaptic pools and to actin may be important for STOP on the of vesicular M. S. D. A. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, F. Full Text Full Text PDF PubMed Scopus Google Scholar, S. Neurosci. PubMed Scopus Google Scholar, M.J. Cell Dev. Biol. PubMed Scopus Google Scholar, T. S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). Additionally, there is evidence for a of actin during synaptic potentiation, both in the and post-synaptic compartments M. S. D. A. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, M.J. Cell Dev. Biol. PubMed Scopus Google Scholar, J. Neurosci. 1998; PubMed Google Scholar, J. Full Text Full Text PDF PubMed Scopus Google Scholar), and we find phosphorylated STOP in the of and post-synaptic protein in differentiated neurons. Finally, microtubule and actin assemblies have been a of in and proteins with have been identified as of the L. S. J. 2004; PubMed Scopus Google Scholar, A. E. A. E. Full Text Full Text PDF PubMed Scopus Google Scholar, C. R. E. Full Text Full Text PDF PubMed Scopus Google Scholar). that STOP protein may function as such in nerve terminals. We D. Proietto and A. Schweitzer for E. Denarier for C. and J. Wehland for in monoclonal antibodies with