©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of Acan125 as a Myosin-I-binding Protein Present with Myosin-I on Cellular Organelles of Acanthamoeba(*)

(Received for publication, June 28, 1995; and in revised form, August 31, 1995)

Pin Xu Anita S. Zot Henry G. Zot (§)

From the Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9040

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have discovered the first protein to bind to a nonfilamentous myosin, aside from actin. This protein, Acan125, is a 125-kDa protein from Acanthamoeba that associates with the SH3 domain of Acanthamoeba myosin-IC and not the SH3 domain of human fodrin. Antibodies raised against Acan125 recognize a single protein of 125 kDa from a whole cell lysate of Acanthamoeba; antibodies to myosin-I (M1.7 and M1.8) do not recognize Acan125 on the same blot. Double labeling of Acanthamoeba show Acan125 and myosin-I to be present on the same intracellular organelle, most likely amoebastomes. Immunoprecipitation with either anti-myosin-I or anti-Acan125 antibodies coprecipitates both Acan125 and myosin-I from a lysate of Acanthamoeba, demonstrating that Acan125 interacts with native myosin-I.


INTRODUCTION

Binding through the Src homology domain, SH3,^1 is a recognized means of linking signal transduction proteins(1, 2, 3) , but a function has not been ascribed to the SH3 domain of the cytoskeletal protein myosin-I. The isoforms of myosin-I that contain an SH3 domain include myosin-Is from Acanthamoeba(4) , Dictyostelium(5) , Saccharomyces(6) , rat(7) , and human(8) . In the known myosin-I sequences, the SH3 domain invariably resides in tandem with one or two proline-rich domains(5, 9) ; a proline-rich sequence of 3BP1 has been identified as a motif that binds to the SH3 domain of Abl (10) . An interaction has not been demonstrated between SH3 and proline-rich domains of myosin-I, but proline-rich domains of myosin-I have been shown to interact with actin(11, 12, 13) . These results suggested to us that the SH3 domain of myosin-I might be available for interaction with another protein.

The proposal of proteins that interact with myosin-I is rooted in efforts to reconcile reconstitution results with cellular localization studies. In vitro binding (11, 14, 15) and motility (16) assays are consistent with myosin-I acting mechanically on the surface of any membrane containing the ubiquitous phospholipid phosphatidylserine. But immunostaining shows myosin-I to be excluded from most cell membranes and to be concentrated at the leading edges of migrating cells (17, 18) and on selected organelles(19) . The contractile vacuole of Acanthamoeba has been demonstrated to selectively bind the myosin-IC isoform(20) . Myosin-IA and myosin-IB were found, using immunogold, to be associated along one side of fractionated membranes(21) , as though bound to proteins.

Myosin-I could associate with other proteins on membrane surfaces via interactions with SH3. SH3 domains have been shown to mediate specific associations between SH3-containing proteins and various binding partners, including phosphatidylinositol 3-kinase(22, 23, 24, 25) , p22, and p47(26, 27, 28) , and dynamin(29, 30, 31) . In each of these studies, bacterially expressed fusion proteins of SH3 domains were used as affinity ligands to selectively extract the binding partner from a cell lysate. Selectivity may be dictated by the structures of both the SH3 domain and its binding partner(32, 33) . Thus, binding partners for myosin-I might be identified by their association with the SH3 domain of myosin-I.

We report here one protein from Acanthamoeba, Acan125, which binds to the SH3 domain of myosin-IC and colocalizes with myosin-I on cellular organelles.


EXPERIMENTAL PROCEDURES

Preparation of GST Fusion Proteins

GST (glutathione S-transferase) fusion protein constructs were prepared from polymerase chain reaction products of SH3 domains of Acanthamoeba myosin-IC and human nonerythroid spectrin (fodrin). The DNA corresponding to amino acids 981-1031 of Acanthamoeba myosin-IC was amplified from the plasmid p4.5L (gift from T. D. Pollard, Johns Hopkins University). The polymerase chain reaction primers were 5`-CCGGATCCGCGCGTGCGCTGTA (sense) and 5`-GATGAATTCGACGTAGGA (antisense), which included BamHI and EcoRI sites in the sense and antisense primers, respectively. We subcloned the product into Bluescript (Stratagene, La Jolla, CA) and verified the nucleotide sequence. The bona fide DNA was subcloned directly into the BamHI and EcoRI cloning sites of the bacterial expression vector pGEX-2TK (Pharmacia, Piscatawa, NJ). We received the sequence verified DNA for the SH3 domain of human nonerythroid spectrin (fodrin) corresponding to residues 974-1030 in the pGEX vector (gift from J. P. Albanesi, University of Texas Southwestern Medical Center). Both constructs and the empty vector were transformed into Escherichia coli DH5alpha cells for expression.

GST fusion proteins were expressed in bacteria and purified by affinity chromatography on glutathione-agarose (Pharmacia Biotech Inc.). The protein was eluted with glutathione, dialyzed, and stored at 4 °C with NaN(3) added. Proteins were stable as determined by SDS-PAGE during the time of the experiments.

Affinity Chromatography of Acanthamoeba Lysate

Acanthamoeba (2 g) were harvested, resuspended in 2 ml of 0.5 times TBS (1 times TBS: 25 mM Tris-HCl, pH 7.4, 3.7 mM KCl, 138 mM NaCl) supplemented with 1 mM phenylmethylsulfonyl fluoride, 100 µg/ml leupeptin, 10 µg/ml pepstatin A, 100 units/ml aprotinin, 1 mg/ml diisopropyl fluorophosphate, and 1 mM dithiothreitol, and lysed with 10 strokes in a Dounce homogenizer. The lysate was cleared by centrifugation at 400,000 times g for 10 min and the supernatant passed through a 0.45-µm filter. A 0.5-ml volume of the filtrate was mixed with 0.2 ml of glutathione beads coupled with 250 µg of fusion protein for 15 min at 4 °C. The beads were washed five times with 1.5 ml of 0.5 times TBS and then eluted with 0.2 ml of 5 times TBS. The proteins were separated on 10% SDS-PAGE and stained with Coomassie Blue.

Antibodies

Polyclonal antibodies were raised against Acan125 in rabbits. Rabbits were injected with 10-30 µg of Acan125 cut from an SDS-PAGE gel (8 times 1 times 0.1 cm) and mixed 1:1 with TiterMax adjuvant (Vaxcel, Norcross, GA). The rabbits were boosted twice with the same antigen and once with the excised Acan125 gel band in phosphate-buffered saline (12 mM NaH(2)PO(4), pH 7.4, 138 mM NaCl, 2.7 mM KCl). Control serum was collected before immunizing the rabbit.

Antibodies to myosin were obtained from other laboratories. From T. D. Pollard (Johns Hopkins University), we received mouse monoclonal antibodies, M1.7 and M1.8, to Acanthamoeba myosin-I, and M2.42, to Acanthamoeba myosin-II. From I. C. Baines (National Institutes of Health), we received rabbit polyclonal antibodies to Acanthamoeba myosin-IC. These antibodies have been characterized elsewhere(20, 34) .

Specificity of the anti-Acan125 antibodies was determined by Western blot. A whole cell lysate of Acanthamoeba was separated on SDS-PAGE and a strip of the gel was cut and stained with Coomassie Blue. The remainder of the gel was transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA) and used for a Surf Blot (Idea Scientific, Minneapolis, MN) in which sealed wells of solution containing the antibodies overlay the filter. This creates lanes for antibody reactivity on a continuous blotting surface and eliminates the problems associated with aligning strips cut from a blot. Peroxidase coupled secondary antibodies to mouse or rabbit IgG (American Qualex, La Mirada, CA) were added to the wells, and the peroxidase reaction was developed on the whole blotting surface with chemiluminescent reagent (ECL, Amersham Corp.).

Immunoprecipitations of Acan125 and Myosin-I

Lysate (0.7 ml) of Acanthamoeba (described above) was mixed with 0.7 ml of 0.5 times TBS and with 20 µl of protein A beads alone (Pharmacia Biotech Inc. and Sigma) or an equal volume of protein A beads coupled with 7 µg of IgG. The suspensions were incubated 60 min at 4 °C with constant gentle mixing. The beads were recovered by gentle centrifugation (82 times g for 1 min) and washed five times with 1 ml of 0.5 times TBS. Proteins were eluted from the beads in 100 µl of 5 times TBS, separated on SDS-PAGE, and transferred to a PVDF membrane. The blots were incubated with the primary antibodies indicated and developed using peroxidase-coupled secondary antibodies and chemiluminescent reagent.

Immunostaining of Acanthamoeba

M1.7 and anti-Acan125 immune serum in phosphate-buffered saline were directly labeled with fluorescent dyes(35) . M1.7 was conjugated with tetramethylrhodamine 6-isothiocyanate (Molecular Probes, Eugene, OR) and purified on G-25 (A/A = 0.40), and anti-Acan125 immune serum was conjugated with fluorescein isothiocyanate (Molecular Probes) and purified on protein A (A/A = 0.12). Acanthamoeba castellanii, Neff strain (American Type Culture Collection, Rockville, MD), were grown in normal liquid culture on polystyrene and split 12-24 h prior to staining. Cells (2-5 times 10^5) were seeded on polylysine-coated coverslips and allowed to attach 60 min in culture medium. One-step fixation was used(19) ; coverslips were immersed in 1% paraformaldehyde and methanol at -20 °C and incubated 5 min. Nonspecific binding was blocked with 1% bovine serum albumin in TBS. For staining, coverslips were incubated 60 min in 110 µl containing 1.4 µg of rhodamine-labeled M1.7, 78 µg of fluorescein labeled anti-Acan125 IgG, and 1% bovine serum albumin in TBS. Fluorescence was observed with a Zeiss Axiovert microscope outfitted with 100X oil immersion lens and standard filter sets for fluorescein and rhodamine. Raw images were captured with a CCD camera, digitized, and stored on disk. Digital images were processed with Adobe Photoshop only to match backgrounds in the two channels and then printed directly by dye sublimation.


RESULTS AND DISCUSSION

To isolate SH3-binding proteins, affinity beads was prepared with the ligand being the SH3 domain of Acanthamoeba myosin-IC (SH3) expressed as a fusion protein of GST. The fusion proteins GST, GST-SH3, or GST-SH3 (human fodrin SH3-negative control) were immobilized on glutathione beads and then mixed with a lysate of Acanthamoeba; best results were obtained using the lysate clarified by centrifugation at 400,000 times g. High salt was used to elute amoeba proteins without disrupting the association between fusion proteins and the beads. The same proteins that eluted from all beads containing fusion proteins were shown to be nonspecifically bound to the beads alone (Fig. 1). Specifically bound proteins were detected exclusively in the high salt wash from the beads containing GST-SH3 (Fig. 1, a-d). Subsequent release of all proteins from the beads with SDS revealed that the same amount of fusion protein was bound to all beads and that no other proteins were specifically associated with SH3 (data not shown). Thus, four proteins, Acan125, (^2)Acan62, Acan55, and Acan47 (Fig. 1), are reversibly associated with SH3.


Figure 1: Specific association of Acan125 with the SH3 domain of Acanthamoeba myosin-IC. Each lane represents a separate binding experiment and shows the proteins that eluted from glutathione beads containing the following fusion proteins: lane 1, none; lane 2, GST; lane 3, GST-SH3; lane 4, GST-SH3. a-d, proteins, Acan125, Acan62, Acan55, and Acan47, that specifically bind to SH3.



Acan125 was selected for further study because it was the least likely to be a proteolytic fragment, the most abundant, and the most well separated from other proteins on the gel. A large preparation yielded 400 µg of the Acan125, which was sufficient for the production of antibodies and the attainment of microsequence data.

Given that the SH3 of Src binds a proline-rich motif (10) and that a proline-rich region is present in the sequence of SH3-containing isoforms of myosin-I(5) , the SH3 domain of myosin-IC could interact with the proline-rich region of another, possibly uncharacterized, myosin-I. To determine if Acan125 is a myosin-I, we transferred Acan125 to a PVDF membrane for microsequence determination. Attempts to obtain sequence directly failed, indicating that the N terminus is blocked. The bound Acan125 was digested with endoproteinase lysC and the peptides separated by high performance liquid chromatography. Several peptide sequences were obtained (data not shown) and searches of protein data bases (PIR 42 and Swiss-Prot 30) did not reveal a match to a known myosin-I.

Polyclonal antibodies were raised in rabbits immunized with Acan125 that was excised from SDS-PAGE. A single protein of 125 kDa was recognized on a Western blot of Acanthamoeba whole cell lysate using anti-Acan125 antibodies (Fig. 2); immune serum, protein A purified immune IgG, and blot purified antibodies gave identical results. On the same blot, mouse monoclonal antibodies to myosin-I (M1.7 and M1.8) recognized multiple bands of proteins (Fig. 2). Previously, M1.7 was shown to react with myosin-IA, myosin-IC, and myosin-II; M1.8 was shown to react with the same proteins plus myosin-IB(34) . The M1.7- and M1.8-reactive proteins on the blot of Acanthamoeba lysate (Fig. 2) correlate with the sizes of myosins, 130-190 kDa. The fact that the broad spectrum myosin antibodies M1.7 and M1.8 recognize proteins larger than the anti-Acan125-reactive protein is consistent with the assertion that Acan125 is not a myosin-I.


Figure 2: Western blot of Acanthamoeba lysate showing the specificity of antibodies used. Lane 1, Coomassie Blue-stained gel showing proteins before transfer; lane 2, M1.8-reactive proteins; lane 3, M1.7-reactive proteins; lane 4, Anti-Acan125-reactive protein. The blotting surface between lanes 2 and 4 is continuous, thereby demonstrating that the anti-Acan125-reactive protein does not comigrate with the anti-myosin-I-reactive proteins.



To demonstrate that Acan125 interacts with myosin-I, we immunoprecipitated the complex from a lysate of Acanthamoeba using M1.7 and M1.8. The myosin-II antibody M2.42 was used as a control. Immunoprecipitations with M1.7 and M1.8, but not with M2.42, showed a single band of reactivity to anti-Acan125 antibodies on a Western blot (Fig. 3A). Thus, Acan125 is precipitated specifically by myosin-I antibodies, indicating that a direct association between the two proteins is likely.


Figure 3: Immunoprecipitations showing that Acan125 and myosin-I form a complex. Western blot A, separate immunoprecipitations were performed using M1.8 (lane 2), M1.7 (lane 3), M2.42 (lane 4), or no antibody (lane 5). A standard of Acan125 protein was included on the same blot (lane 1). The entire blot was probed with anti-Acan125 antibodies. Western blot B, separate immunoprecipitations were performed using no antibody (lane a), preimmune serum (lane b), anti-Acan125 antibodies (lane c), and M1.7 (lane d). The entire blot was probed with M1.7.



To verify Acan125 association with myosin-I, myosin-I was coprecipitated with Acan125 antibodies from a lysate of Acanthamoeba. Anti-Acan125 and preimmune sera were used to form immunoprecipitates, but only the anti-Acan125 antibodies coprecipitated proteins that reacted with M1.7. The blot shows at least two bands of M1.7 reactivity in the immunoprecipitation (Fig. 3B), indicating that Acan125 interacts with more than one isoform of myosin-I. We identified one isoform, myosin-IC, in the lower band in Fig. 3B using antibodies specific for myosin-IC (data not shown); we have not yet identified a specific isoform of myosin-I in the upper band. The ability of anti-Acan125 antibodies to precipitate myosin-I isoforms from a soluble lysate suggests that complexes of Acan125 and myosin-I exist in Acanthamoeba.

To assess potential interactions in vivo, we stained Acanthamoeba cells for both myosin-I and Acan125. Myosin-I was detected using M1.7 labeled with rhodamine, and Acan125 was detected using protein A-purified anti-Acan125 antibodies labeled with fluorescein. Rhodamine-labeled M1.7 staining of Acanthamoeba was characterized by diffuse fluorescence throughout the cytoplasm excluding the interior of vacuoles, by intense fluorescence in the nuclear region, and by occasional intense fluorescence circumscribing a single round structure (Fig. 4A). The same round structure was stained by fluorescein-labeled anti-Acan125 antibodies in double-labeled cells (Fig. 4B).


Figure 4: Double-stained Acanthamoeba show the colocalization of myosin-I- and Acan125-reactive proteins. The same field shows M1.7 staining in the rhodamine channel (panel A) and anti-Acan125 staining in the fluorescein channel (panel B). Marked are amoebastomes (arrow), defined by M1.7 staining, and one example of nucleoplasmic staining (n).



Staining in the nuclear region by rhodamine-labeled M1.7 was observed in all cells (one cell is marked and two cells are unmarked in Fig. 4A). This fluorescence arises from intense staining of the nucleoplasm that surrounds a single large unstained nucleolus (visible in the unmarked cells in Fig. 4A). The nucleoplasmic staining is absent from identically prepared cells stained with unlabeled M1.7 and a labeled secondary antibody. Although the nucleoplasmic staining appears to be an artifact of the rhodamine-labeled M1.7, this provides a convenient internal control for the present experiment. The nucleoplasm is not stained with anti-Acan125 antibodies (Fig. 4B); however, the round structures in Fig. 4are double-stained, revealing the colocalization of myosin-I and Acan125.

These round structures appear to be similar to structures described earlier as vacuoles in Acanthamoeba stained with M1.7(19) . But a more recent exhaustive study (^3)indicates that M1.7 stains myosin-I on tubular structures known as amoebastomes, which appear similar to vacuoles in cross-section. All amoebastomes that stained with M1.7 also stained with anti-Acan125 antibodies in our experiments. No structures were observed to be stained exclusively by anti-Acan125 antibodies, and we found no more than a single organelle per cell to be stained by both antibodies. Colocalization of Acan125- and myosin-I-reactive proteins on M1.7-stained organelles, which are probably amoebastomes, further indicates the formation of a complex of myosin-I and Acan125 in the cell.

Acan125 is presently the only protein aside from actin known to interact with myosin-I. However, we think it is likely that others will be identified including Acan62, Acan55, and Acan47, which bind myosin-I SH3 and do not react with anti-Acan125 antibodies on a blot. The SH3 domain of myosin-I may regulate or target myosin-I's interactions with these proteins in response to cellular signals. The presence of Acan125 and myosin-I in the high speed supernatant suggest that they are both present in the cytoplasm, and the immunoprecipitation results demonstrate that Acan125 and myosin-I are competent to form a complex. The complex may be recruited to organelle surfaces where attachment could be mediated by either Acan125 or myosin-I. A role for the complex will become clearer when a function can be ascribed to Acan125.


FOOTNOTES

*
This work was supported by Grant MCB-9205344 from the National Science Foundation and Grant 92-1571 from the American Heart Association National Center. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept of Physiology, UT Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9040. Tel.: 214-648-7224; Fax: 214-648-8685; zot@utsw.swmed.edu.

(^1)
The abbreviations used are: SH3, Src homology 3; GST, glutathione S-transferase; TBS, Tris-buffered saline; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride.

(^2)
In a preliminary report (Zot, A. S., and Zot, H. G.(1994) Mol. Biol. Cell5, 162A), Acan125 was incorrectly estimated to be 150 kDa.

(^3)
S. K. Doberstein, I. C. Baines, E. D. Korn, and T. D. Pollard, personal communication.


ACKNOWLEDGEMENTS

We thank Dr. Tom Pollard and his laboratory for making their clones and antibodies available and for critical reading of the manuscript, Drs. Joe Albanesi and Ofer Riezes for supplying the human fodrin SH3, and Dr. Ivan Baines for making antibodies available and for assistance leading to the identification of amoebastomes in our preparation. We also thank Teresa Davison for expert technical assistance.


REFERENCES

  1. Pawson, T., and Gish, G. D. (1992) Cell 71, 359-362 [Medline] [Order article via Infotrieve]
  2. Schlessinger, J. (1994) Curr. Opin. Genet. Dev. 4, 25-30 [Medline] [Order article via Infotrieve]
  3. Feller, S. M., Ren, R., Hanafusa, H., and Baltimore, D. (1994) Trends Biochem. Sci. 19, 453-458 [CrossRef][Medline] [Order article via Infotrieve]
  4. Rodaway, A. R., Sternberg, M. J. E., and Bentley, D. L. (1989) Nature 342, 624 [Medline] [Order article via Infotrieve]
  5. Pollard, T. D., Doberstein, S. K., and Zot, H. G. (1991) Annu. Rev. Physiol. 53, 653-681 [CrossRef][Medline] [Order article via Infotrieve]
  6. Goodson, H. V., and Spudich, J. A. (1995) Cell Motil. Cytoskel. 30, 73-84 [Medline] [Order article via Infotrieve]
  7. Stöffler, H.-E., Ruppert, C., Reinhard, R., and Bähler, M. (1995) J. Cell Biol. 129, 819-830 [Abstract]
  8. Bement, W. M., Wirth, J. A., and Mooseker, M. S. (1994) J. Cell Biol. 243, 356-363
  9. Hammer, J. A., III (1994) J. Musc. Res. Cell Motil. 15, 1-10 [Medline] [Order article via Infotrieve]
  10. Ren, R., Mayer, B. J., Cicchetti, P., and Baltimore, D. (1993) Science 259, 1157-1161 [Medline] [Order article via Infotrieve]
  11. Doberstein, S. K., and Pollard, T. D. (1992) J. Cell Biol. 117, 1241-1249 [Abstract]
  12. Rosenfeld, S. S., and Rener, B. (1994) Biochemistry 33, 2322-2328 [Medline] [Order article via Infotrieve]
  13. Jung, G., and Hammer, J. A., III (1994) FEBS Lett. 342, 197-202 [CrossRef][Medline] [Order article via Infotrieve]
  14. Adams, R. A., and Pollard, T. D. (1989) Nature 340, 565-588 [CrossRef][Medline] [Order article via Infotrieve]
  15. Hayden, S. M., Wolenski, J. S., and Mooseker, M. S. (1990) J. Cell Biol. 111, 443-451 [Abstract]
  16. Zot, H. G., Doberstein, S. K., and Pollard, T. D. (1992) J. Cell Biol. 116, 367-376 [Abstract]
  17. Fukui, Y., Lynch, T. J., Brzeska, H., and Korn, E. D. (1989) Nature 341, 328-331 [CrossRef][Medline] [Order article via Infotrieve]
  18. Conrad, P. A., Giuliano, K. A., Fisher, G., Collins, K., Matsudaira, P. T., and Taylor, D. L. (1993) J. Cell Biol. 120, 1381-1391 [Abstract]
  19. Yonemura, S., and Pollard, T. D. (1992) J. Cell Sci. 102, 629-642 [Abstract]
  20. Baines, I. C., and Korn, E. D. (1989) J. Cell Biol. 111, 1895-1904 [Abstract]
  21. Baines, I. C., Brzeska, H., and Korn, E. D. (1992) J. Cell Biol. 119, 1193-1203 [Abstract]
  22. Prasad, K. V., Janssen, O., Kapeller, R., Raab, M., Cantley, L. C., and Rudd, C. E. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 7366-7370 [Abstract]
  23. Liu, X., Marengere, L. E. M., Koch, C. A., and Pawson, T. (1993) Mol. Cell. Biol. 13, 5225-5232 [Abstract]
  24. Pleiman, C. M., Hertz, W. M., and Cambier, J. C. (1994) Science 263, 1609-1612 [Medline] [Order article via Infotrieve]
  25. Kapeller, R., Prasad, K. V. S., Janssen, O., Hou, W., Schaffhausen, B. S., Rudd, C. E., and Cantley, L. C. (1994) J. Biol. Chem. 269, 1927-1933 [Abstract/Free Full Text]
  26. Sumimoto, H., Kage, Y., Nunoi, H., Sasaki, H., Nose, T., Fukumaki, Y., Ohno, M., Minakami, S., and Takishige, K. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5345-5349 [Abstract]
  27. Finan, P., Shimizu, Y., Gout, I., Hsuan, J., Truong, O., Butcher, C., Bennett, P., Waterfield, M. D., and Kellie, S. (1994) J. Biol. Chem. 269, 13752-13755 [Abstract/Free Full Text]
  28. Leto, T. L., Adams, A. G., and De Mendez, I. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10650-10654 [Abstract/Free Full Text]
  29. Gout, I., Dhand, R., Hiles, I. D., Fry, I. J., Panayotou, G., Das, P., Truong, O., Totty, N. F., Hsuan, J., Booker, G. W., Campbell, I. D., and Waterfield, M. D. (1993) Cell 75, 25-36 [Medline] [Order article via Infotrieve]
  30. Miki, H., Miura, K., Matuoka, K., Nakata, T., Hirokawa, N., Orita, S., Kaibuchi, K., Takai, Y., and Takenawa, T. (1994) J. Biol. Chem. 269, 5489-5492 [Abstract/Free Full Text]
  31. Seedof, K., Kostka, G., Lammers, R., Bashkin, P., Daly, R., Burgess, W. H., van der Bliek, A. M., Schlessinger, J., and Ullrich, A. (1994) J. Biol. Chem. 269, 16009-16014 [Abstract/Free Full Text]
  32. Lim, W. A., Richards, F. M., and Fox, R. D. (1994) Nature 372, 375-379 [CrossRef][Medline] [Order article via Infotrieve]
  33. Koyama, S, Yu, H., Dalgarno, D. C., Shin, T. B., Zydowsky, L. D., and Schreiber, S. L. (1993) Cell 72, 945-952 [Medline] [Order article via Infotrieve]
  34. Hagan, S. J., Kiehart, D. P., Kaiser, D. A., and Pollard, T. D. (1986) J. Cell Biol. 103, 2121-2128 [Abstract]
  35. Harlow, E., and Lane, D. (1988) Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.