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J. Biol. Chem., Vol. 275, Issue 44, 34017-34020, November 3, 2000

This Article Abstract Full Text (PDF) All Versions of this Article: 275/44/34017    most recent C000429200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lai, M. M. Articles by Snyder, S. H. Search for Related Content PubMed PubMed Citation Articles by Lai, M. M. Articles by Snyder, S. H. Social Bookmarking                      What's this?

ACCELERATED PUBLICATION
The Calcineurin-binding Protein Cain Is a Negative Regulator of Synaptic Vesicle Endocytosis^*

Michael M. Lai, Hongbo R. Luo, Patrick E. Burnett, Jenny J. Hong, and Solomon H. Snyder^§¶

From the Departments of  Neuroscience, ^§ Pharmacology and Molecular Sciences, and ^¶ Psychiatry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Received for publication, July 3, 2000, and in revised form, July 21, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

During neurotransmitter release, exocytosed neurotransmitter vesicles are recycled by endocytosis, which involves the assembly of a complex of endocytic proteins. Assembly of endocytic proteins into a functional complex depends on their dephosphorylation by calcineurin, a calcium-sensitive protein phosphatase and the inhibitory target of immunosuppressive drugs cyclosporin A and FK506. Cain is a recently identified protein inhibitor of calcineurin. We now provide evidence that cain is a component of the endocytic protein complex. The proline-rich region of cain forms a stable association with the SH3 domain of amphiphysin 1. Using a transferrin uptake assay, we found that overexpression of cain in HEK293 cells blocks endocytosis as potently as expression of a dominant negative dynamin 1 construct. The use of other calcineurin inhibitors such as cyclosporin A and FK506 also blocks endocytosis. Since binding of cain to amphiphysin 1 does not affect amphiphysin's interaction with other endocytic proteins, our results suggest that cain negatively regulates synaptic vesicle endocytosis by inhibiting calcineurin activity, rather than sterically interfering with the assembly of the endocytic protein complex.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The release of neurotransmitters from nerve terminals employs a calcium-dependent exocytotic process involving a group of proteins that interact in a calcium-dependent fashion (1, 2). The burst of calcium that initiates exocytosis also leads to synaptic vesicle recycling by endocytosis of the released vesicles and is mediated by a unique complex of endocytic proteins, which includes amphiphysin, synaptojanin, dynamin 1, clathrin, and clathrin adapters (3-5). Assembly of the endocytic proteins into a functional complex depends on their dephosphorylation (6, 7). We have recently demonstrated that the calcium-sensitive phosphatase calcineurin is physically linked to the endocytic proteins via its interaction with dynamin 1, the GTPase component of the synaptic vesicle coat complex (8). The calcium-dependent nature of the calcineurin-dynamin 1 interaction acts as a calcium sensor for endocytosis, which is initiated by calcineurin-mediated dephosphorylation of endocytic proteins.

Recently, we and others (9, 10) identified a novel protein designated cain (calcineurin inhibitor) or cabin (calcineurin-binding protein), which binds to and inhibits calcineurin. Cain/cabin was initially cloned from brain and lymphocyte libraries but has subsequently been found in a broad range of tissues. In T cells, cabin has been shown to regulate T cell receptor (TCR)-induced apoptosis through its interaction with the transcription factor myocyte enhancer factor 2 (MEF2) (11). However, cain's role in the nervous system has not been clearly elucidated. Given its large size (240 kDa) and modular organization, we had hypothesized that cain may function as a scaffolding protein linking calcineurin to a variety of target proteins. We now show that cain binds to amphiphysin 1 in the endocytic complex and, in providing localized inhibition of calcineurin, serves as a physiologic negative regulator of endocytosis.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Co-immunoprecipitation of Cain and Amphiphysin 1-- Adult rat brain was homogenized in lysis buffer (50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 2 mM CaCl₂, 2 mM MgCl₂, 0.2% Triton X-100, 0.5 mM -mercaptoethanol, 5 µg/ml aprotinin, 1 µg/ml leupeptin, 6 µg/ml chymostatin, 0.7 µg/ml pepstatin, 1 mM phenylmethylsulfonlyl fluoride) and centrifuged at 20,000 × g for 20 min to remove insoluble materials. The resulting lysate was first precleared with rabbit IgG and protein G-agarose for 1 h 4 °C and then incubated with cain antiserum and protein G-agarose for 2 h at 4 °C. Immunoprecipitated proteins were washed four times with lysis buffer, eluted in SDS sample buffer, and analyzed by immunoblotting with anti-amphiphysin 1 antibody.

Cain-Amphiphysin 1 Binding Experiments-- GST¹-amphiphysin and GST-amphSH3 proteins were expressed in Escherichia coli cells and purified with glutathione-agarose as per the manufacturer's recommendations (Amersham Pharmacia Biotech). HEK293 cells were transfected with the calcium phosphate precipitate method with myc-tagged cain variants as described previously (12). After 24-48 h, cells were harvested and lysed in lysis buffer. Equal aliquots of the lysate were incubated with GST-amphiphysin or GST-amphSH3 for 1 h at 4 °C and then washed four times with lysis buffer. Bound proteins were analyzed by SDS-polyacrylamide gel electrophoresis followed by immunoblotting with anti-myc antibody.

Preparation of ELISA Plates-- ELISA plates were prepared according to Smythe et al. (13) and Carter et al. (14). Briefly, goat anti-human transferrin antibody (Sigma) was plated onto Immuno-plate (Pierce) at a 1:10,000 dilution in 200 µl of 50 mM Na₂HCO₃ (pH 9.6). Plates were incubated at 4 °C overnight, washed three times with PBS, and then incubated for 30 min at 37 °C in ELISA blocking buffer (1% Triton, 0.1% SDS, 0.2% BSA, 50 mM NaCl, 1 mM EDTA, 10 mM Tris (pH 7.4)). Plates were stored in blocking buffer at 4 °C.

Receptor-mediated Endocytosis of Transferrin-- HEK293 cells were transiently transfected with the indicated expression constructs as before. After 48 h, cells were serum starved for 2 h and then subjected to the ELISA-based transferrin uptake assay described by Smythe et al. (13) and Carter et al. (14). Cells from each 10-cm plate were harvested at room temperature in PBS containing 5 mM EDTA, washed twice with PBS, and resuspended in 1 ml of ice-cold PBS containing 1 mM CaCl₂, 1 mM MgCl₂, 5 mM glucose, 0.2% BSA, and 3 µg/ml biotin-labeled transferrin (Sigma). Transferrin internalization was performed by incubating the cell suspension at 37 °C for indicated time. The reactions were stopped by returning the tubes back on ice. The cells were then pelleted and resuspended in 100 µl of PBS containing 1 mM CaCl₂, 1 mM MgCl₂, 5 mM glucose, 0.2% BSA, and 50 µg/ml avidin (Sigma). After 1-h incubation at 4 °C, Biocytin (Sigma) was added to a final concentration of 50 µg/ml, and agitation was continued for 10 min. The cells were lysed with ELISA blocking buffer and plated on the ELISA plates. Total cell-associated transferrin was determined from the lysate of untransfected cells subjected to the same buffer additions without avidin. The plates were incubated at 37 °C for 3 h and then washed twice with blocking buffer. After adding 0.5 µg/ml streptavidin-horseradish peroxidase (Roche Molecular Biochemicals), the plates were incubated for an additional 60 min at room temperature and washed three times with blocking buffer. The bounded horseradish peroxidase concentrations were measured at A₄₀₅ in the presence of one-step ABTS substrate (Pierce).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Cain is a 240-kDa protein that binds calcineurin through a 38-amino acid region near its C-terminal tail (Fig. 1A). It is hypothesized that the rest of cain, which contains several potential protein-protein interacting domains, allows cain to act as a scaffolding protein linking calcineurin to other target proteins in the cell. The mid-portion of cain possesses a proline-rich domain (amino acids 1748-1876) whose properties closely resemble those of other proteins that bind with some selectivity to the SH3 domain of the endocytic protein amphiphysin 1 (15). Accordingly, we wondered whether cain might directly interact with amphiphysin 1 and thereby "deliver" localized inhibition of calcineurin activity within the synaptic endocytic complex. To test this hypothesis, we conducted immunoprecipitation experiments in whole rat brain lysates using anti-cain antibody. Western blot analysis of the immunoprecipitate with an antibody to amphiphysin 1 reveals a robust interaction between cain and amphiphysin 1, which is not evident with preimmune serum (Fig. 1B).

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Cain associates with amphiphysin 1. A, schematic representation of cain protein. The protein-protein interacting domains are as labeled: CC, coiled-coil region; PRD, proline-rich domain; and CBD, calcineurin-binding domain. B, rat brain lysate was immunoprecipitated with cain antiserum or preimmune serum. Immunoblotting of the precipitate with anti-amphiphysin 1 antibody revealed co-immunoprecipitation of cain and amphiphysin 1. C, rat brain lysate was immunoprecipitated with cain antiserum or preimmune serum and then immunoblotted with the indicated antibody. Cain co-immunoprecipitates with amphiphysin 1, dynamin 1, and adaptin, but not Rap2.

The SH3 domain of amphiphysin 1 has been shown previously to bind the proline-rich region of dynamin 1 (16). We wondered whether the cain-amphiphysin 1 interaction could co-exist with the dynamin 1-amphiphysin 1 interaction or whether the binding of cain to amphiphysin 1 displaces dynamin 1 from the SH3 domain. To address this issue, we checked the cain immunoprecipitate for other co-immunoprecipitated proteins by Western immunoblotting. We found that amphiphysin 1, dynamin 1, and adaptin all co-immunoprecipitate with cain; whereas an unrelated GTP-binding protein Rap2 does not interact with any member of this complex (Fig. 1C). Our results demonstrate that both cain and dynamin 1 can simultaneously bind the SH3 domain of amphiphysin 1. Furthermore, the stable association between cain and amphiphysin 1 suggests that cain is an integral component of the synaptic endocytic protein complex.

To further clarify the domains of cain and amphiphysin that interact, we conducted binding experiments utilizing various truncations of myc-cain and GST-amphiphysin 1 (Fig. 2A). GST-amphiphysin 1 binds to full-length myc-cain and to its proline-rich domain but not to any other portion of cain. To confirm the involvement of the SH3 domain of amphiphysin 1, we examined the ability of the isolated SH3 domain of GST-amphiphysin 1 to bind to myc-cain (Fig. 2B). The SH3 domain of amphiphysin 1 behaves identically as the full-length protein in binding to both the full-length and proline-rich domain of myc-cain, suggesting that it contains all the necessary sequence for binding cain. Thus, interactions between the two proteins are mediated by the proline-rich domain of cain and the SH3 domain of amphiphysin 1. 

View larger version (30K): [in this window] [in a new window]   Fig. 2.   The proline-rich region of cain binds the SH3 domain of amphiphysin 1. A, HEK293 cell lysates expressing the indicated myc-tagged cain constructs were incubated with GST-amphiphysin 1 resin. Bound proteins were visualized by immunoblotting with anti-myc antibody. Only the full-length cain construct (cainFL) and the construct containing the proline-rich region (cainPR) bind GST-amphiphysin 1. The corresponding amino acids of the constructs are as follows: cainFL, aa 1-2182; cainN, aa 1-726; cain2, aa 721-1077; cain3, aa 1070-1230; cain4, aa 1225-1753; cainPR, aa 1748-1876; cainCBD, aa 1880-2173. B, HEK293 cell lysates containing myc-cainFL, myc-cainN, and myc-cainPR constructs were incubated with the GST-amphSH3 resin, which contains the SH3 domain of amphiphysin 1. Only the full-length cain protein and the proline-rich region of cain bind GST-amphSH3.

In earlier studies, we showed that calcineurin activates synaptic vesicle endocytic process by dephosphorylating endocytic proteins (7). Since cain is a physiologic inhibitor of calcineurin, we hypothesize that it may deactivate endocytosis. Accordingly, we monitored the uptake of biotinylated transferrin in HEK293 cells, a procedure that has been shown to reflect endocytic events (17, 18). As a positive control, we transfected a dominant negative construct of dynamin 1 (dynamin K44E), in which a lysine critical for the GTPase activity of dynamin has been mutated to glutamic acid. Expression of dynamin K44E has been shown to alter clathrin distribution and block transferrin uptake (19). In our system, transferrin internalization in HEK293 cells transfected with vector alone increases linearly for 5 min and then plateaus with a modest decline at 25 min. During the linear portion of the time course, transfection with the mutant dynamin K44E construct reduces transferrin uptake by about 34-38%. Since the transfection efficiency is only about 50% (data not shown), this reflects a 70% reduction in endocytosis and is consistent with the known role of dynamin 1 in mediating transferrin endocytosis. These results resemble the findings of Schmid and associates (17), who found a 40% reduction of transferrin uptake by the same mutant dynamin construct transiently transfected into HeLa cells at 25-60% efficiency. Mutant dynamin K44E stably transfected into HeLa cells, with presumed 100% efficiency, reduced transferrin uptake about 75% (18). Overexpression of cain in our system reduces endocytosis to about the same extent as the expression of dominant negative dynamin 1 (Fig. 3).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Cain inhibits transferrin endocytosis. The kinetics of transferrin endocytosis by HEK293 cells transfected with the indicated constructs were analyzed as described under "Experimental Procedures." Expression of cain blocks transferrin endocytosis as potently as the expression of the dominant negative dynamin K44E construct. The results are the mean of four independent experiments. The mean and S.E. values at the 5-min time point are as follows: vector alone, 80 ± 2.9; cain, 60 ± 3.2; dynamin K44E, 61 ± 2.3.

If endocytosis reflects dephosphorylation by calcineurin, then inhibitors of calcineurin other than cain should also reduce endocytosis. The immunosuppressant drugs cyclosporin A and FK506 are known inhibitors of calcineurin (20, 21). Treatment with cyclosporin A or FK506 reduces endocytosis to the same extent as does cain overexpression (Table I). Rapamycin is an immunosuppressant drug that binds with high affinity to FKBP12, but the rapamycin-FKBP12 complex does not affect calcineurin (22-24). Accordingly, rapamycin can be employed as an antagonist of the pharmacological actions of FK506. In our system, rapamycin prevents the ability of FK506 to reduce endocytosis but has no effect on endocytosis by itself. Likewise, GPI1046, another compound that binds FKBP12 but does not inhibit calcineurin (25), has no effect on endocytosis. Taken together, inhibition of calcineurin activity, whether endogenously by cain or exogenously by cyclosporin A or FK506, leads to reduction of endocytosis.

De Camilli and associates (7) showed that all of the proteins of the synaptic vesicle endocytic complex, including clathrin, adaptin, dynamin 1, amphiphysin 1, amphiphysin 2, and synaptojanin, must be dephosphorylated to assemble into an active complex (6). Previously, we demonstrated that calcineurin is the calcium sensor that initiates these events and mediates the dephosphorylation of these proteins (8). In the present study, we extend this model by showing that cain can bind directly to amphiphysin 1 as well as to calcineurin and therefore functions as a physiologic inhibitor of endocytosis (Fig. 4). Our co-immunoprecipitation experiments establish that cain binds to calcineurin and to amphiphysin 1 simultaneously, which is consistent with different domains of cain involved in two separate protein-protein interactions. Specifically, the proline-rich domain of cain binds to amphiphysin 1, while its C terminus binds to calcineurin. In our earlier study, we showed that calcineurin binds to dynamin 1 directly and only indirectly to other proteins of the endocytic complex (7). Since binding of cain to amphiphysin 1 does not affect amphiphysin's interaction with other endocytic proteins, we postulate that cain terminates synaptic vesicle endocytosis by inhibiting calcineurin activity, rather than sterically interfering with the endocytic protein complex assembly.

View larger version (29K): [in this window] [in a new window]   Fig. 4.   Proposed model of the role of cain in negatively regulating endocytosis. Please refer to "Results and Discussion" for a detailed discussion of the proposed model. Briefly, the assembly of endocytic proteins adaptin (adp), amphiphysin 1 (amph1), amphiphysin 2 (amph2), clathrin (cla), dynamin 1 (dyn1), and synaptojanin (syj) into a functional complex is driven by calcineurin (Cn)-mediated dephosphorylation. In the presence of increased intracellular calcium concentration, active calcineurin is delivered to this complex of proteins via its interaction with dynamin 1. Once the endocytic coat complex is fully assembled, cain, which is stably associated with amphiphysin 1, promotes the rephosphorylation and dissociation of endocytic proteins by inactivating calcineurin. In this regard, cain may work in concert with cellular kinases to negatively regulate the assembly of endocytic protein complex.

The immunosuppressive drugs cyclosporin A and FK506 achieve their therapeutic effect of preventing host-versus-graft rejections by inhibiting calcineurin activity. In the present study, we show that these drugs also impair synaptic vesicle endocytosis by mimicking the calcineurin inhibitory action of cain. Post-transplantation patients receiving cyclosporin A or FK506 commonly exhibit a variety of neurologic symptoms including tremors, seizures, and leukoencephalopathy (26-28). Future works may elucidate a mechanistic connection between these clinical observations and calcineurin's role in synaptic vesicle endocytosis.

	ACKNOWLEDGEMENTS

We thank Alicia Ruggiero and Keqiang Ye for technical assistance and Levente Egry and Regis Kelly for invaluable discussions.

	FOOTNOTES

^* This work was supported by United States Public Health Service Grant MH-18501 from the National Institute of Mental Health, Research Scientist Award DA-00074 (to S. H. S.) from National Institute on Drug Abuse, and Training Grant GM-07309 (to M. M. L.) from NIGMS, National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 410-955-3024; Fax: 410-614-6249; E-mail: ssnyder@jhmi.edu.

Published, JBC Papers in Press, August 7, 2000, DOI 10.1074/jbc.C000429200

	ABBREVIATIONS

The abbreviations used are: GST, glutathione S-transferase; ELISA, enzyme-linked immunosorbent assay; BSA, bovine serum albumin; PBS, phosphate-buffered saline; aa, amino acid(s).

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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This Article Abstract Full Text (PDF) All Versions of this Article: 275/44/34017    most recent C000429200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lai, M. M. Articles by Snyder, S. H. Search for Related Content PubMed PubMed Citation Articles by Lai, M. M. Articles by Snyder, S. H. Social Bookmarking                      What's this?