- Email: [email protected]
The RIN Family of Ras Effectors
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Clark, G. J., Cox, A. D., Graham, S. M., and Der, C. J. (1995). Biological assays for Ras transformation. Methods Enzymol. 255, 395–412. Mitin, N. Y., Ramocki, M. B., Zullo, A. J., Der, C. J., Konieczny, S. F., and Taparowsky, E. J. (2004). Identification and characterization of rain, a novel Ras‐interacting protein with a unique subcellular localization. J. Biol. Chem. 279, 22353–22361. Morgenstern, J. P., and Land, H. (1990). Advanced mammalian gene transfer: High titre retroviral vectors with multiple drug selection markers and a complementary helper‐free packaging cell line. Nucleic Acids Res. 18, 3587–3596. St Croix, B., Rago, C., Velculescu, V., Traverso, G., Romans, K. E., Montgomery, E., Lal, A., Riggins, G. J., Lengauer, C., Vogelstein, B., and Kinzler, K. W. (2000). Genes expressed in human tumor endothelium. Science 289, 1197–1202.
[28] The RIN Family of Ras Effectors By JOANNE M. BLISS, BYRAPPA VENKATESH , and JOHN COLICELLI Abstract
The human RIN1 gene was first identified as a cDNA fragment that interfered with RAS‐induced phenotypes in the yeast Saccharomyces cerevisiae. Subsequent analysis of full‐length RIN1 clones showed that the protein product of this gene is a downstream effector of RAS and binds with high affinity and specificity to activated HRAS. Two downstream RIN1 effector pathways have been described. The first involves direct activation of RAB5‐mediated endocytosis. The second involves direct activation of ABL tyrosine kinase activity. Importantly, each of these distinct RIN1 functions is enhanced by activated RAS, suggesting that RIN1 represents a unique class of RAS effector connected to two independent signaling pathways. In this chapter, we summarize our assays and approaches for evaluating the biochemistry and biology of RIN1. Introduction
The human RIN1 gene was first identified as a cDNA fragment that interfered with RAS‐induced phenotypes in the yeast Saccharomyces cerevisiae (Colicelli et al., 1991). Subsequent analysis of full‐length RIN1 clones showed that the protein product of this gene binds with high affinity and specificity to activated HRAS (Han and Colicelli, 1995; Wang et al., 2002). Two downstream RIN1 effector pathways have been described. The first involves direct activation of RAB5‐mediated endocytosis (Tall et al., 2001). The second involves direct activation of ABL tyrosine kinase activity METHODS IN ENZYMOLOGY, VOL. 407 Copyright 2006, Elsevier Inc. All rights reserved.
0076-6879/06 $35.00 DOI: 10.1016/S0076-6879(05)07028-X
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(Hu et al., 2005). Importantly, each of these distinct RIN1 functions is enhanced by activated RAS, suggesting that RIN1 represents a unique class of RAS effector connected to two independent signaling pathways. The RIN1 protein has four domains defined by sequence alignment and functional studies (Fig. 1). The amino terminal half of RIN1 includes a SRC homology 2 (SH2) domain that binds to the cytoplasmic regions of several receptor tyrosine kinases (Barbieri et al., 2003). Also in the amino terminus of RIN1 are tyrosine phosphorylation substrates and proline‐rich domains that together facilitate stable binding to the SH2 and SH3 domains, respectively, of ABL family tyrosine kinases (Hu et al., 2005). In the carboxy terminal section of RIN1 are found a RAS association (RA) domain and a guanine nucleotide exchange factor (GEF) domain of the subclass most related to the vacuolar protein sorting 9 (VPS9) protein. In addition to RIN1, there are two other members of the RIN family in mammals. RIN2 and RIN3 show the same domain structure as RIN1 with regions of strong sequence conservation (Fig. 1). In the SH2 domain, however, some family members carry a substitution at the arginine within the FLVR motif that is critical for phosphotyrosine binding. Specifically, human RIN2 has a histidine, whereas mouse and rat RIN3 have cysteines in this position, raising the possibility that these isoforms do not bind receptor tyrosine kinases in the same way as has been characterized for RIN1. The RA domains of RIN1, RIN2, and RIN3 are well conserved. Although the RAS binding characteristics have been best characterized for
FIG. 1. RIN family proteins. Graphic representation of the human RIN1, RIN2, and RIN3 proteins (products of the ENSEMBL genes ENSG00000174791, ENSG00000132669, and ENSG00000100599). Single horizontal lines in RIN1 and RIN2 indicate gaps introduced by sequence alignment (ClustalW) with RIN3. Shaded regions (gray and black) represent regions with sequence identities of 39% or greater when comparing RIN1 to RIN2 and RIN2 to RIN3. Black boxes indicate established functional domains (SH2, SRC Homology 2; VPS9, yeast Vacuolar Sorting Protein 9 related with GEF activity; RA, RAS association). The ABL binding domain (ABD, aa 1–295) and RAS binding domain (RBD, aa 296–727) of RIN1 are labeled.
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RIN1, basic RAS binding has been demonstrated for RIN2 and RIN3 as well (Rodriguez‐Viciana et al., 2004). RAB5 guanine nucleotide exchange factor activity has been characterized in RIN1, RIN2, and RIN3 (Kajiho et al., 2003; Saito et al., 2002; Tall et al., 2001) and localized to the Vps9‐type GEF domain. In addition to these functional domains, all of the RIN proteins include proline‐rich motifs with potential SH3‐binding properties. A RIN family conserved domain of unknown function, sometimes called an RH domain (Kajiho et al., 2003; Saito et al., 2002), is found upstream of the GEF domain. Finally, two phosphorylation events have been characterized for RIN1. The first involves phosphorylation by PKD of serine 351 (human) and promotes binding to 14–3‐3 proteins (Wang et al., 2002). This site is conserved in mammalian RIN1 orthologs but not in RIN2 or RIN3. The second established phosphorylation is by ABL tyrosine kinases on tyrosine 36 (human) and perhaps other tyrosines. The preferred ABL substrate sequence, YxxP, appears three times in RIN1 (including Y36), and these motifs are conserved in rat and mouse. However, this sequence is absent in RIN2 and appears only once in RIN3, and, at present, there is no evidence that these proteins are substrates for ABL or any other tyrosine kinase. Assays of RIN Protein Function
RIN1 has two characterized biochemical functions: activation of RAB5 proteins (Tall et al., 2001) and activation of ABL proteins (Hu et al., 2005). The guanine nucleotide exchange properties, encoded within the GEF (VPS9) domain of RIN1 as well as RIN2 and RIN3, promote GDP release and GTP loading on RAB5 proteins, leading to increased levels of receptor endocytosis. There are multiple assays to evaluate this function. Measurements of GDP Release and GTP Loading on RAB5 Proteins Several established assays of guanine nucleotide exchange factor function on small GTPase substrates have been adapted to RAB5 activity measurements. The GEF function of RIN1 was first demonstrated using in vitro assays of radioactively labeled GDP release and GTP loading (Tall et al., 2001). Similar techniques were used to demonstrate the GEF activity of RIN2 and RIN3 (Kajiho et al., 2003; Saito et al., 2002). Importantly, the GEF activity of RIN1 was enhanced by the addition of HRAS‐GTP, demonstrating that this function is part of the RAS effector response. Measurement of GTP/GDP Ratios for RAB5 Proteins It should also be possible to evaluate RIN function through determinations of guanine nucleotide occupation of RAB5 proteins isolated from cell extracts. This approach allows for the evaluation of controlled stimulatory
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effects (e.g., growth factor treatment) on RIN engagement and RAB5 activation. As with other small GTPases, guanine nucleotide occupation can be evaluated by chromatographic methods after immunoprecipitation of the protein (Balch et al., 1995). Alternately, it is possible to preferentially purify GTP‐bound RAB5 using a fusion protein that includes the RAB5 binding domain of the effector protein RABEP1 (a.k.a. rabaptin‐5) fused to a GST domain (Brown et al., 2005). Isolated RAB5‐RAB5 can then be detected by immunoblot and normalized to total RAB5. Measurement of Receptor Endocytosis Levels Because RAB5 proteins facilitate receptor endocytosis (Segev, 2001), enhanced rates of endocytosis can serve as an indirect measure of RIN protein activity. Internalization of radioactive EGF was used to demonstrate that RIN1 promotes endocytosis in NR6 cells (Barbieri et al., 2003; Tall et al., 2001). An internal deletion of RIN1 that disrupted the GEF domain was shown to inhibit endocytosis, perhaps through a dominant negative effect. As a corollary to the increased receptor internalization resulting from RIN1 overexpression, it is also possible to detect decreases in EGF receptor downstream signaling after activation of RAB5 through RIN1 overexpression (Barbieri et al., 2004). A second function of RIN1 is the stimulation of ABL tyrosine kinase activity, although it should be noted that this property has not yet been demonstrated for RIN2 and RIN3 proteins. There are multiple assays that can be used to determine relative levels of ABL stimulation. The tyrosine kinases ABL1 (a.k.a. c‐Abl) and ABL2 (a.k.a. Arg) are normally maintained in low‐activity conformations (reviewed in Hantschel and Superti‐Furga [2004] ). RIN1 binds to the SH3 domain of ABL through a proline‐rich sequence, and the subsequent tyrosine phosphorylation of RIN1 then promotes binding to the SH2 domain of ABL. This divalent interaction seems to activate the tyrosine kinase activity of ABL proteins. The stimulatory effect of RIN1 has been studied primarily using ABL2, which, like RIN1, localizes to the cell cytoplasm and membrane surfaces (by contrast, much of ABL1 is localized in the nucleus). Kinase assays can be carried out using ABL2 protein immunoprecipitated from cell extracts and an ABL substrate peptide or a purified ABL substrate such as CRK (Hu et al., 2005). Alternately, in vitro kinase assays can be performed using ABL2 purified from insect cells together with purified CRK and the controlled addition of the ABL binding fragment of RIN1. Assays using all purified components show lower levels of RIN1‐mediated stimulation than cell extract immunoprecipitates, suggesting that as yet unidentified cellular factors may contribute to this signaling pathway.
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Immunoprecipitation‐Kinase Assay Because endogenous ABL2 and RIN1 levels are quite low in most cell lines, this assay is best performed with cells that overexpress an ABL2 construct with or without a RIN1 construct. The most pronounced effects are seen with a RIN1 construct that is missing the RAS‐binding domain (RBD), which normally acts as an autorepressor, but retains the ABL‐ binding domain (ABD). HEK 293T cells are transfected with pcDNA3‐ ABL2‐Flag and vector alone or pcDNA3‐RIN1‐ABD. Two days after transfection, cells are lysed in nonionic detergent buffer, and ABL2 protein is immunoprecipitated using anti‐Flag agarose beads (Sigma‐Aldrich). This IP material is washed twice with lysis buffer and once with kinase buffer (10 mM Tris‐HCl, pH 7.4, 10 mM MgCl2 100 mM NaCl, 1 mM DTT, 1 mM Na3VO4). ABL2 levels can be normalized by immunoblot and densitometry of cell lysates using anti‐Flag (Sigma). Biotinylated ABL1/2 substrate peptide, bio‐AQDVYDVPPAKKK (10 M) is mixed with IP material and kinase buffer to a final volume of 50 l. The reaction is initiated by addition of ATP (10 M) with ‐32P‐ATP (1 Ci) and allowed to proceed for 10 min at 30 . Reactions are stopped with 250 mM EDTA. The peptide is then bound to avidin sepharose beads (Pierce), washed three times with PBS þ 20 M ATP. The incorporated 32P is measured by scintillation counter. Alternately, the assay can be performed using purified GST‐ CRK and the product detected by immunoblot with anti‐CRK‐pTyr221. It is advisable to carry out a 1‐h time course experiment to confirm that the conditions used are within the linear range for enzyme kinetics. To validate the ABL dependence of the kinase assay results, a control sample with 10 M of the ABL inhibitor STI571 can be carried out. A 10 mM stock solution of STI571 can be made by dissolving the contents of a 100‐mg capsule of Gleevec (Novartis) in 17 ml water. Inert material is then removed by centrifugation. ABL‐Mediated Changes in Cell Function Cytoplasmic ABL proteins are regulators of cytoskeletal actin remodeling (reviewed in Hernandez et al. [2004] ). Overexpression of ABL induces membrane microspikes in some cell types (Woodring et al., 2002, 2004). In fibroblast cells, overexpression of the ABL activator RIN1 produces a similar phenotype (Hu et al., 2005). ABL kinase activity has also been shown to inhibit cell migration. More specifically, cell migration is enhanced by deletion of ABL genes (Kain and Klemke, 2001), by addition of the ABL inhibitor STI571 (Frasca et al., 2001; Hu et al., 2005), or by deletion or knockdown of RIN1 (Hu et al., 2005). A quantifiable cell migration assay can, therefore, be used to indirectly assess RIN1 function.
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Transwell migration assays can be performed using a variety of cell lines including the human mammary epithelial cell derived MCF10A. These cells are grown in DMEM/F12 plus hEGF (20 ng/ml), hydrocortisone (500 ng/ml), insulin (10 g/ml), cholera toxin (100 ng/ml), and horse serum (5%). Migration assays were performed in Boyden chambers (Costar, Inc.) with membranes undercoated with 10 M fibronectin. Cells suspended in serum‐free medium are added to the upper chamber and allowed to migrate for 4–16 h at 37 toward the lower chamber, which contains 50 ng/ml HGF (Sigma‐Aldrich). Cells that migrate through the filter are fixed with paraformaldehyde, stained with crystal violet, and counted. In this assay, cells pretreated with RIN1‐directed siRNA showed increased motility (Hu et al., 2005), whereas cells overexpressing RIN1 showed reduced motility (Hu and Colicelli, unpublished data). Immunological Reagents for RIN Protein Analysis
RIN proteins can be detected using antibodies from several commercial sources as well as antibodies developed by individual researcher laboratories. Table I provides a list of antibodies to RIN family proteins and their demonstrated applications (applications not listed may simply not have been examined). Most antibodies to RIN1 show specificity toward the species of antigen to which they were raised (human and mouse RIN1 sequences are 78% identical). RIN Family Evolution
The family of RIN genes is well conserved among mammals, including human, rat, and mouse (Fig. 2). Interestingly, in more basal vertebrates represented by ray‐finned fishes such as zebrafish (Danio rerio) and puffer fish (Takifugu rubripes), there is evidence of RIN2 gene duplication. This duplication is probably the result of the whole‐genome duplication in the ray‐finned fish lineage (Christoffels et al., 2004). Presumably, additional copies of RIN1 and RIN3 were lost subsequent to genome duplication. The resulting duplicate fish RIN2a and RIN2b genes either share the functions of their ancestral precursor (subfunctionalization) or have taken on specialized functions (neofunctionalization) that allow for their continued stability in this lineage. The fruit fly (Drosophila melanogaster) has a single RIN‐related gene named SPRINT (Szabo et al., 2001), which may represent the progenitor of the vertebrate RIN family. Although SPRINT remains largely uncharacterized, it is noteworthy that this gene is expressed in central nervous system neurons (similar to RIN1, see following) as well as in the developing midgut and amnio serosa (Szabo et al., 2001).
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RIN family of Ras effectors TABLE I ANTIBODIES TO RIN PROTEINS Antibody
Specificitya
Applicationsa
Source/Reference
IB, IP IF, IHC IB, IP, IHC IB IB IB, IP IB, IP IB, IP, IHC IB, IP
Transduction Laboratories
Rabbit anti‐RIN1 (h‐Cterm)
Human
Mouse anti‐RIN1 (h‐Cterm) Goat anti‐RIN1 (h‐Nterm) Goat anti‐RIN1 (h‐Cterm) Rabbit anti‐RIN1 (h‐Cterm) Rabbit anti‐RIN1 (h‐Nterm) Rabbit anti‐RIN1 (m‐Cterm) Mouse anti‐RIN1 (m‐Cterm)
Human Human Human Human Human Mouse Mouse
Rabbit anti‐RIN1pY36 b Rabbit anti‐RIN1 splice jct.c
Human & Mouse Human
IB, IP
Rabbit anti‐RIN2d
Human
IB
Polyclonal anti‐RIN3e
Human
IB
IB, IP
Transduction Laboratories Santa Cruz Biotechnology Santa Cruz Biotechnology Han and Colicelli, 1995 Hu et al., 2005 Hu et al., 2005 Colicelli laboratory, unpublished Hu et al., 2005 Colicelli laboratory, unpublished Colicelli laboratory, unpublished Kajiho et al., 2003
Each antibody is listed with a brief description of the antigen to which it was raised (h, human, m, mouse; Cterm, carboxy terminal fragment including the RA and VPS9 domains, Nterm, amino terminal fragment including the SH2 and ABL‐binding domains). Antibody applications include immunoblot (IB), immunoprecipitation (IP), immunoflurosecence (IF), and immunohistochemistry (IHC). a Based on personal experience, published data, and manufacturer claims. b Immunogenic and affinity purification peptides described in reference. c Raised against the human RIN1 splice junction peptide CVSPKRLELEQVRQ. d Raised against uncharacterized fragments of human RIN2 purified from bacteria. e No details on immunogen provided.
No RIN orthologs are found in the nematode worm (C. elegans) or unicellular eukaryotes. However, the pairing of RA and GEF domains, as seen in the amino terminus of RIN proteins, is observed in these organisms, suggesting an important role for the physical connection of these functional domains. RIN Genes Have Distinct Patterns of Expression
It is not yet clear to what extent RIN1, RIN2, and RIN3 carry out distinct functions. A report that RIN3 and RIN2, but not RIN1, associate with BIN1 (a.k.a. amphiphysin II) provides one indication that there may be isoform‐specific signaling pathways (Kajiho et al., 2003). Some inferences may also be drawn from sequence comparisons among family members, such as the multiple well‐conserved ABL phosphorylation sites (YxxP) in RIN1 and the abundance of potential SH3 binding sites (PxxP)
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FIG. 2. RIN family evolution. Unrooted tree derived from comparisons of predicted amino acid sequences of the indicated RIN genes using the program ClustalW. Human (Homo sapiens, Hs); rat (Rattus norvegicus, Rn); mouse (Mus musculus, Mm); zebrafish (Danio rerio, Dr); puffer fish (Takifugu rubripes, Tr), fly (Drosophila melanogaster, Dm). The predicted fly sequence used in this analysis was trimmed to remove several large unaligned regions.
in RIN3. Another indication of distinct functions may be gleaned from observed differences in tissue‐type expression patterns. Each of the RIN family genes seems to have a distinct pattern of expression in mammalian tissues. The highest levels of RIN1 expression in mouse are found in forebrain structures (cortex, hippocampus, amygdala, striatum, and olfactory bulb). This was determined by directed studies (Dhaka et al., 2003) and from use of available databases (GNF SymAtlas 1.0.3, symatlas.gnf.org/SymAtlas). In addition, RIN1 expression was noted in mouse testis (Dhaka et al., 2003), in mammary epithelial cells (Hu et al., 2005), and in some hematopoietic cells (Minh Thai and John Colicelli, unpublished). Limited expression studies using human tissue (Dhaka et al. [2003], GNF SymAtlas, and unpublished data) have been generally consistent with the findings reported for mouse. In many cell lines examined (HeLa, MCF7, LNCAP, JURKAT, and K562), RIN1 is found at low to moderate levels (SymAtlas). RIN1 protein can be detected by direct
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immunoblot of brain extract (60 g total protein) using appropriate antibodies (see ‘‘Reagents’’). From moderately expressing tissues (e.g., mammary epithelial cells) and cell lines (e.g., HeLa) detection of RIN1 protein requires concentration by immunoprecipitation before immunoblot analysis. Endogenous RIN1 is undetectable in the widely used HEK 293T and NIH 3T3 cell lines (Hu et al., 2005). Expression of RIN2 seems to be fairly broad in mouse tissues (SymAtlas), with some possible elevation in lung, bronchial epithelial cells, placenta, uterus, pancreatic islets, thyroid, and hematopoietic cells. In a survey of human tissues, lung, heart, and kidney showed elevated expression (Saito et al., 2002). Most human cell lines tested (HEK 293T, MCF7, LNCAP, JURKAT, K562, and PANC1) showed relatively low expression (SymAtlas). RIN3 expression in mouse is strongly elevated in hematopoietic tissues and cells including bone marrow and isolated B and T cells, as well as some cell lines derived from these sources (SymAtlas). Elevated RIN3 expression levels in human peripheral blood and spleen (Kajiho et al., 2003) are consistent with this pattern and suggest a specialized function in hematopoietic cells. Significant but lower levels of RIN3 message (mouse and human) were found in most other tissues examined. Many human cell lines (HEK 293T, MCF7, LNCAP, JURKAT, K562, PANC1, and HeLa) showed moderate levels of RIN3 expression (SymAtlas). Evidence of splice variants of all three RIN genes comes from cDNA cloning and Northern blot analysis (Han et al., 1997; Kajiho et al., 2003; Saito et al., 2002). In the case of RIN1, evidence of multiple protein products has also been seen (unpublished data). Acknowledgments The authors would like to acknowledge Ms Tay Boon Hui for technical assistance in cloning and sequencing of Takifugu rubripes cDNAs and Minh Thai for development and characterization of several unpublished antibodies. J. C. is supported by NIH grant NS046787 and DoD Breast Cancer Research Program grant W81XWH0410443.
References Balch, W. E., Der, C. J., and Hall, A. (eds.) (1995). ‘‘Small GTPases and Their Regulators.’’ Academic Press, New York. Barbieri, M. A., Fernandez‐Pol, S., Hunker, C., Horazdovsky, B. H., and Stahl, P. D. (2004). Role of rab5 in EGF receptor‐mediated signal transduction. Eur. J. Cell Biol. 83, 305–314. Barbieri, M. A., Kong, C., Chen, P. I., Horazdovsky, B. F., and Stahl, P. D. (2003). The SRC homology 2 domain of Rin1 mediates its binding to the epidermal growth factor receptor and regulates receptor endocytosis. J. Biol. Chem. 278, 32027–32036. Brown, T. C., Tran, I. C., Backos, D. S., and Esteban, J. A. (2005). NMDA receptor‐ dependent activation of the small GTPase Rab5 drives the removal of synaptic AMPA receptors during hippocampal LTD. Neuron 45, 81–94.
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Christoffels, A., Koh, E. G., Chia, J. M., Brenner, S., Aparicio, S., and Venkatesh, B. (2004). Fugu genome analysis provides evidence for a whole‐genome duplication early during the evolution of ray‐finned fishes. Mol. Biol. Evol. 21, 1146–1151. Colicelli, J., Nicolette, C., Birchmeier, C., Rodgers, L., Riggs, M., and Wigler, M. (1991). Expression of three mammalian cDNAs that interfere with RAS function in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 88, 2913–2917. Dhaka, A., Costa, R. M., Hu, H., Irvin, D. K., Patel, A., Kornblum, H. I., Silva, A. J., O’Dell, T. J., and Colicelli, J. (2003). The Ras effector Rin1 modulates the formation of aversive memories. J. Neurosci. 23, 748–757. Frasca, F., Vigneri, P., Vella, V., Vigneri, R., and Wang, J. Y. (2001). Tyrosine kinase inhibitor STI571 enhances thyroid cancer cell motile response to Hepatocyte Growth Factor. Oncogene 20, 3845–3856. Han, L., and Colicelli, J. (1995). A human protein selected for interference with Ras function interacts directly with Ras and competes with Raf1. Mol. Cell. Biol. 15, 1318–1323. Han, L., Wong, D., Dhaka, A., Afar, D., White, M., Xie, W., Herschman, H., Witte, O., and Colicelli, J. (1997). Protein binding and signaling properties of RIN1 suggest a unique effector function. Proc. Natl. Acad. Sci. USA 94, 4954–4959. Hantschel, O., and Superti‐Furga, G. (2004). Regulation of the c‐Abl and Bcr‐Abl tyrosine kinases. Nat. Rev. Mol. Cell. Biol. 5, 33–44. Hernandez, S. E., Krishnaswami, M., Miller, A. L., and Koleske, A. J. (2004). How do Abl family kinases regulate cell shape and movement? Trends Cell Biol. 14, 36–44. Hu, H., Bliss, J. M., Wang, Y., and Colicelli, J. (2005). RIN1 is an ABL tyrosine kinase activator and a regulator of epithelial cell adhesion and migration. Curr. Biol. 15, 815–823. Kain, K. H., and Klemke, R. L. (2001). Inhibition of cell migration by Abl family tyrosine kinases through uncoupling of Crk‐CAS complexes. J. Biol. Chem. 276, 16185–16192. Kajiho, H., Saito, K., Tsujita, K., Kontani, K., Araki, Y., Kurosu, H., and Katada, T. (2003). RIN3: A novel Rab5 GEF interacting with amphiphysin II involved in the early endocytic pathway. J. Cell Sci. 116, 4159–4168. Rodriguez‐Viciana, P., Sabatier, C., and McCormick, F. (2004). Signaling specificity by Ras family GTPases is determined by the full spectrum of effectors they regulate. Mol. Cell. Biol. 24, 4943–4954. Saito, K., Murai, J., Kajiho, H., Kontani, K., Kurosu, H., and Katada, T. (2002). A novel binding protein composed of homophilic tetramer exhibits unique properties for the small GTPase Rab5. J. Biol. Chem. 277, 3412–3418. Segev, N. (2001). Ypt/rab gtpases: Regulators of protein trafficking. Sci. STKE 18, RE11. Szabo, K., Jekely, G., and Rorth, P. (2001). Cloning and expression of sprint, a Drosophila homologue of RIN1. Mech. Dev. 101, 259–262. Tall, G. G., Barbieri, M. A., Stahl, P. D., and Horazdovsky, B. F. (2001). Ras‐activated endocytosis is mediated by the Rab5 guanine nucleotide exchange activity of RIN1. Dev. Cell 1, 73–82. Wang, Y., Waldron, R. T., Dhaka, A., Patel, A., Riley, M. M., Rozengurt, E., and Colicelli, J. (2002). The RAS effector RIN1 directly competes with RAF and is regulated by 14‐ 3–3 proteins. Mol. Cell. Biol. 22, 916–926. Woodring, P. J., Litwack, E. D., O’Leary, D. D., Lucero, G. R., Wang, J. Y., and Hunter, T. (2002). Modulation of the F‐actin cytoskeleton by c‐Abl tyrosine kinase in cell spreading and neurite extension. J. Cell Biol. 156, 879–892. Woodring, P. J., Meisenhelder, J., Johnson, S. A., Zhou, G. L., Field, J., Shah, K., Bladt, F., Pawson, T., Niki, M., Pandolfi, P. P., Wang, J. Y., and Hunter, T. (2004). c‐Abl phosphorylates Dok1 to promote filopodia during cell spreading. J. Cell Biol. 165, 493–503.
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Clark, G. J., Cox, A. D., Graham, S. M., and Der, C. J. (1995). Biological assays for Ras transformation. Methods Enzymol. 255, 395–412. Mitin, N. Y., Ramocki, M. B., Zullo, A. J., Der, C. J., Konieczny, S. F., and Taparowsky, E. J. (2004). Identification and characterization of rain, a novel Ras‐interacting protein with a unique subcellular localization. J. Biol. Chem. 279, 22353–22361. Morgenstern, J. P., and Land, H. (1990). Advanced mammalian gene transfer: High titre retroviral vectors with multiple drug selection markers and a complementary helper‐free packaging cell line. Nucleic Acids Res. 18, 3587–3596. St Croix, B., Rago, C., Velculescu, V., Traverso, G., Romans, K. E., Montgomery, E., Lal, A., Riggins, G. J., Lengauer, C., Vogelstein, B., and Kinzler, K. W. (2000). Genes expressed in human tumor endothelium. Science 289, 1197–1202.
[28] The RIN Family of Ras Effectors By JOANNE M. BLISS, BYRAPPA VENKATESH , and JOHN COLICELLI Abstract
The human RIN1 gene was first identified as a cDNA fragment that interfered with RAS‐induced phenotypes in the yeast Saccharomyces cerevisiae. Subsequent analysis of full‐length RIN1 clones showed that the protein product of this gene is a downstream effector of RAS and binds with high affinity and specificity to activated HRAS. Two downstream RIN1 effector pathways have been described. The first involves direct activation of RAB5‐mediated endocytosis. The second involves direct activation of ABL tyrosine kinase activity. Importantly, each of these distinct RIN1 functions is enhanced by activated RAS, suggesting that RIN1 represents a unique class of RAS effector connected to two independent signaling pathways. In this chapter, we summarize our assays and approaches for evaluating the biochemistry and biology of RIN1. Introduction
The human RIN1 gene was first identified as a cDNA fragment that interfered with RAS‐induced phenotypes in the yeast Saccharomyces cerevisiae (Colicelli et al., 1991). Subsequent analysis of full‐length RIN1 clones showed that the protein product of this gene binds with high affinity and specificity to activated HRAS (Han and Colicelli, 1995; Wang et al., 2002). Two downstream RIN1 effector pathways have been described. The first involves direct activation of RAB5‐mediated endocytosis (Tall et al., 2001). The second involves direct activation of ABL tyrosine kinase activity METHODS IN ENZYMOLOGY, VOL. 407 Copyright 2006, Elsevier Inc. All rights reserved.
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(Hu et al., 2005). Importantly, each of these distinct RIN1 functions is enhanced by activated RAS, suggesting that RIN1 represents a unique class of RAS effector connected to two independent signaling pathways. The RIN1 protein has four domains defined by sequence alignment and functional studies (Fig. 1). The amino terminal half of RIN1 includes a SRC homology 2 (SH2) domain that binds to the cytoplasmic regions of several receptor tyrosine kinases (Barbieri et al., 2003). Also in the amino terminus of RIN1 are tyrosine phosphorylation substrates and proline‐rich domains that together facilitate stable binding to the SH2 and SH3 domains, respectively, of ABL family tyrosine kinases (Hu et al., 2005). In the carboxy terminal section of RIN1 are found a RAS association (RA) domain and a guanine nucleotide exchange factor (GEF) domain of the subclass most related to the vacuolar protein sorting 9 (VPS9) protein. In addition to RIN1, there are two other members of the RIN family in mammals. RIN2 and RIN3 show the same domain structure as RIN1 with regions of strong sequence conservation (Fig. 1). In the SH2 domain, however, some family members carry a substitution at the arginine within the FLVR motif that is critical for phosphotyrosine binding. Specifically, human RIN2 has a histidine, whereas mouse and rat RIN3 have cysteines in this position, raising the possibility that these isoforms do not bind receptor tyrosine kinases in the same way as has been characterized for RIN1. The RA domains of RIN1, RIN2, and RIN3 are well conserved. Although the RAS binding characteristics have been best characterized for
FIG. 1. RIN family proteins. Graphic representation of the human RIN1, RIN2, and RIN3 proteins (products of the ENSEMBL genes ENSG00000174791, ENSG00000132669, and ENSG00000100599). Single horizontal lines in RIN1 and RIN2 indicate gaps introduced by sequence alignment (ClustalW) with RIN3. Shaded regions (gray and black) represent regions with sequence identities of 39% or greater when comparing RIN1 to RIN2 and RIN2 to RIN3. Black boxes indicate established functional domains (SH2, SRC Homology 2; VPS9, yeast Vacuolar Sorting Protein 9 related with GEF activity; RA, RAS association). The ABL binding domain (ABD, aa 1–295) and RAS binding domain (RBD, aa 296–727) of RIN1 are labeled.
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RIN1, basic RAS binding has been demonstrated for RIN2 and RIN3 as well (Rodriguez‐Viciana et al., 2004). RAB5 guanine nucleotide exchange factor activity has been characterized in RIN1, RIN2, and RIN3 (Kajiho et al., 2003; Saito et al., 2002; Tall et al., 2001) and localized to the Vps9‐type GEF domain. In addition to these functional domains, all of the RIN proteins include proline‐rich motifs with potential SH3‐binding properties. A RIN family conserved domain of unknown function, sometimes called an RH domain (Kajiho et al., 2003; Saito et al., 2002), is found upstream of the GEF domain. Finally, two phosphorylation events have been characterized for RIN1. The first involves phosphorylation by PKD of serine 351 (human) and promotes binding to 14–3‐3 proteins (Wang et al., 2002). This site is conserved in mammalian RIN1 orthologs but not in RIN2 or RIN3. The second established phosphorylation is by ABL tyrosine kinases on tyrosine 36 (human) and perhaps other tyrosines. The preferred ABL substrate sequence, YxxP, appears three times in RIN1 (including Y36), and these motifs are conserved in rat and mouse. However, this sequence is absent in RIN2 and appears only once in RIN3, and, at present, there is no evidence that these proteins are substrates for ABL or any other tyrosine kinase. Assays of RIN Protein Function
RIN1 has two characterized biochemical functions: activation of RAB5 proteins (Tall et al., 2001) and activation of ABL proteins (Hu et al., 2005). The guanine nucleotide exchange properties, encoded within the GEF (VPS9) domain of RIN1 as well as RIN2 and RIN3, promote GDP release and GTP loading on RAB5 proteins, leading to increased levels of receptor endocytosis. There are multiple assays to evaluate this function. Measurements of GDP Release and GTP Loading on RAB5 Proteins Several established assays of guanine nucleotide exchange factor function on small GTPase substrates have been adapted to RAB5 activity measurements. The GEF function of RIN1 was first demonstrated using in vitro assays of radioactively labeled GDP release and GTP loading (Tall et al., 2001). Similar techniques were used to demonstrate the GEF activity of RIN2 and RIN3 (Kajiho et al., 2003; Saito et al., 2002). Importantly, the GEF activity of RIN1 was enhanced by the addition of HRAS‐GTP, demonstrating that this function is part of the RAS effector response. Measurement of GTP/GDP Ratios for RAB5 Proteins It should also be possible to evaluate RIN function through determinations of guanine nucleotide occupation of RAB5 proteins isolated from cell extracts. This approach allows for the evaluation of controlled stimulatory
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effects (e.g., growth factor treatment) on RIN engagement and RAB5 activation. As with other small GTPases, guanine nucleotide occupation can be evaluated by chromatographic methods after immunoprecipitation of the protein (Balch et al., 1995). Alternately, it is possible to preferentially purify GTP‐bound RAB5 using a fusion protein that includes the RAB5 binding domain of the effector protein RABEP1 (a.k.a. rabaptin‐5) fused to a GST domain (Brown et al., 2005). Isolated RAB5‐RAB5 can then be detected by immunoblot and normalized to total RAB5. Measurement of Receptor Endocytosis Levels Because RAB5 proteins facilitate receptor endocytosis (Segev, 2001), enhanced rates of endocytosis can serve as an indirect measure of RIN protein activity. Internalization of radioactive EGF was used to demonstrate that RIN1 promotes endocytosis in NR6 cells (Barbieri et al., 2003; Tall et al., 2001). An internal deletion of RIN1 that disrupted the GEF domain was shown to inhibit endocytosis, perhaps through a dominant negative effect. As a corollary to the increased receptor internalization resulting from RIN1 overexpression, it is also possible to detect decreases in EGF receptor downstream signaling after activation of RAB5 through RIN1 overexpression (Barbieri et al., 2004). A second function of RIN1 is the stimulation of ABL tyrosine kinase activity, although it should be noted that this property has not yet been demonstrated for RIN2 and RIN3 proteins. There are multiple assays that can be used to determine relative levels of ABL stimulation. The tyrosine kinases ABL1 (a.k.a. c‐Abl) and ABL2 (a.k.a. Arg) are normally maintained in low‐activity conformations (reviewed in Hantschel and Superti‐Furga [2004] ). RIN1 binds to the SH3 domain of ABL through a proline‐rich sequence, and the subsequent tyrosine phosphorylation of RIN1 then promotes binding to the SH2 domain of ABL. This divalent interaction seems to activate the tyrosine kinase activity of ABL proteins. The stimulatory effect of RIN1 has been studied primarily using ABL2, which, like RIN1, localizes to the cell cytoplasm and membrane surfaces (by contrast, much of ABL1 is localized in the nucleus). Kinase assays can be carried out using ABL2 protein immunoprecipitated from cell extracts and an ABL substrate peptide or a purified ABL substrate such as CRK (Hu et al., 2005). Alternately, in vitro kinase assays can be performed using ABL2 purified from insect cells together with purified CRK and the controlled addition of the ABL binding fragment of RIN1. Assays using all purified components show lower levels of RIN1‐mediated stimulation than cell extract immunoprecipitates, suggesting that as yet unidentified cellular factors may contribute to this signaling pathway.
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Immunoprecipitation‐Kinase Assay Because endogenous ABL2 and RIN1 levels are quite low in most cell lines, this assay is best performed with cells that overexpress an ABL2 construct with or without a RIN1 construct. The most pronounced effects are seen with a RIN1 construct that is missing the RAS‐binding domain (RBD), which normally acts as an autorepressor, but retains the ABL‐ binding domain (ABD). HEK 293T cells are transfected with pcDNA3‐ ABL2‐Flag and vector alone or pcDNA3‐RIN1‐ABD. Two days after transfection, cells are lysed in nonionic detergent buffer, and ABL2 protein is immunoprecipitated using anti‐Flag agarose beads (Sigma‐Aldrich). This IP material is washed twice with lysis buffer and once with kinase buffer (10 mM Tris‐HCl, pH 7.4, 10 mM MgCl2 100 mM NaCl, 1 mM DTT, 1 mM Na3VO4). ABL2 levels can be normalized by immunoblot and densitometry of cell lysates using anti‐Flag (Sigma). Biotinylated ABL1/2 substrate peptide, bio‐AQDVYDVPPAKKK (10 M) is mixed with IP material and kinase buffer to a final volume of 50 l. The reaction is initiated by addition of ATP (10 M) with ‐32P‐ATP (1 Ci) and allowed to proceed for 10 min at 30 . Reactions are stopped with 250 mM EDTA. The peptide is then bound to avidin sepharose beads (Pierce), washed three times with PBS þ 20 M ATP. The incorporated 32P is measured by scintillation counter. Alternately, the assay can be performed using purified GST‐ CRK and the product detected by immunoblot with anti‐CRK‐pTyr221. It is advisable to carry out a 1‐h time course experiment to confirm that the conditions used are within the linear range for enzyme kinetics. To validate the ABL dependence of the kinase assay results, a control sample with 10 M of the ABL inhibitor STI571 can be carried out. A 10 mM stock solution of STI571 can be made by dissolving the contents of a 100‐mg capsule of Gleevec (Novartis) in 17 ml water. Inert material is then removed by centrifugation. ABL‐Mediated Changes in Cell Function Cytoplasmic ABL proteins are regulators of cytoskeletal actin remodeling (reviewed in Hernandez et al. [2004] ). Overexpression of ABL induces membrane microspikes in some cell types (Woodring et al., 2002, 2004). In fibroblast cells, overexpression of the ABL activator RIN1 produces a similar phenotype (Hu et al., 2005). ABL kinase activity has also been shown to inhibit cell migration. More specifically, cell migration is enhanced by deletion of ABL genes (Kain and Klemke, 2001), by addition of the ABL inhibitor STI571 (Frasca et al., 2001; Hu et al., 2005), or by deletion or knockdown of RIN1 (Hu et al., 2005). A quantifiable cell migration assay can, therefore, be used to indirectly assess RIN1 function.
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Transwell migration assays can be performed using a variety of cell lines including the human mammary epithelial cell derived MCF10A. These cells are grown in DMEM/F12 plus hEGF (20 ng/ml), hydrocortisone (500 ng/ml), insulin (10 g/ml), cholera toxin (100 ng/ml), and horse serum (5%). Migration assays were performed in Boyden chambers (Costar, Inc.) with membranes undercoated with 10 M fibronectin. Cells suspended in serum‐free medium are added to the upper chamber and allowed to migrate for 4–16 h at 37 toward the lower chamber, which contains 50 ng/ml HGF (Sigma‐Aldrich). Cells that migrate through the filter are fixed with paraformaldehyde, stained with crystal violet, and counted. In this assay, cells pretreated with RIN1‐directed siRNA showed increased motility (Hu et al., 2005), whereas cells overexpressing RIN1 showed reduced motility (Hu and Colicelli, unpublished data). Immunological Reagents for RIN Protein Analysis
RIN proteins can be detected using antibodies from several commercial sources as well as antibodies developed by individual researcher laboratories. Table I provides a list of antibodies to RIN family proteins and their demonstrated applications (applications not listed may simply not have been examined). Most antibodies to RIN1 show specificity toward the species of antigen to which they were raised (human and mouse RIN1 sequences are 78% identical). RIN Family Evolution
The family of RIN genes is well conserved among mammals, including human, rat, and mouse (Fig. 2). Interestingly, in more basal vertebrates represented by ray‐finned fishes such as zebrafish (Danio rerio) and puffer fish (Takifugu rubripes), there is evidence of RIN2 gene duplication. This duplication is probably the result of the whole‐genome duplication in the ray‐finned fish lineage (Christoffels et al., 2004). Presumably, additional copies of RIN1 and RIN3 were lost subsequent to genome duplication. The resulting duplicate fish RIN2a and RIN2b genes either share the functions of their ancestral precursor (subfunctionalization) or have taken on specialized functions (neofunctionalization) that allow for their continued stability in this lineage. The fruit fly (Drosophila melanogaster) has a single RIN‐related gene named SPRINT (Szabo et al., 2001), which may represent the progenitor of the vertebrate RIN family. Although SPRINT remains largely uncharacterized, it is noteworthy that this gene is expressed in central nervous system neurons (similar to RIN1, see following) as well as in the developing midgut and amnio serosa (Szabo et al., 2001).
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RIN family of Ras effectors TABLE I ANTIBODIES TO RIN PROTEINS Antibody
Specificitya
Applicationsa
Source/Reference
IB, IP IF, IHC IB, IP, IHC IB IB IB, IP IB, IP IB, IP, IHC IB, IP
Transduction Laboratories
Rabbit anti‐RIN1 (h‐Cterm)
Human
Mouse anti‐RIN1 (h‐Cterm) Goat anti‐RIN1 (h‐Nterm) Goat anti‐RIN1 (h‐Cterm) Rabbit anti‐RIN1 (h‐Cterm) Rabbit anti‐RIN1 (h‐Nterm) Rabbit anti‐RIN1 (m‐Cterm) Mouse anti‐RIN1 (m‐Cterm)
Human Human Human Human Human Mouse Mouse
Rabbit anti‐RIN1pY36 b Rabbit anti‐RIN1 splice jct.c
Human & Mouse Human
IB, IP
Rabbit anti‐RIN2d
Human
IB
Polyclonal anti‐RIN3e
Human
IB
IB, IP
Transduction Laboratories Santa Cruz Biotechnology Santa Cruz Biotechnology Han and Colicelli, 1995 Hu et al., 2005 Hu et al., 2005 Colicelli laboratory, unpublished Hu et al., 2005 Colicelli laboratory, unpublished Colicelli laboratory, unpublished Kajiho et al., 2003
Each antibody is listed with a brief description of the antigen to which it was raised (h, human, m, mouse; Cterm, carboxy terminal fragment including the RA and VPS9 domains, Nterm, amino terminal fragment including the SH2 and ABL‐binding domains). Antibody applications include immunoblot (IB), immunoprecipitation (IP), immunoflurosecence (IF), and immunohistochemistry (IHC). a Based on personal experience, published data, and manufacturer claims. b Immunogenic and affinity purification peptides described in reference. c Raised against the human RIN1 splice junction peptide CVSPKRLELEQVRQ. d Raised against uncharacterized fragments of human RIN2 purified from bacteria. e No details on immunogen provided.
No RIN orthologs are found in the nematode worm (C. elegans) or unicellular eukaryotes. However, the pairing of RA and GEF domains, as seen in the amino terminus of RIN proteins, is observed in these organisms, suggesting an important role for the physical connection of these functional domains. RIN Genes Have Distinct Patterns of Expression
It is not yet clear to what extent RIN1, RIN2, and RIN3 carry out distinct functions. A report that RIN3 and RIN2, but not RIN1, associate with BIN1 (a.k.a. amphiphysin II) provides one indication that there may be isoform‐specific signaling pathways (Kajiho et al., 2003). Some inferences may also be drawn from sequence comparisons among family members, such as the multiple well‐conserved ABL phosphorylation sites (YxxP) in RIN1 and the abundance of potential SH3 binding sites (PxxP)
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FIG. 2. RIN family evolution. Unrooted tree derived from comparisons of predicted amino acid sequences of the indicated RIN genes using the program ClustalW. Human (Homo sapiens, Hs); rat (Rattus norvegicus, Rn); mouse (Mus musculus, Mm); zebrafish (Danio rerio, Dr); puffer fish (Takifugu rubripes, Tr), fly (Drosophila melanogaster, Dm). The predicted fly sequence used in this analysis was trimmed to remove several large unaligned regions.
in RIN3. Another indication of distinct functions may be gleaned from observed differences in tissue‐type expression patterns. Each of the RIN family genes seems to have a distinct pattern of expression in mammalian tissues. The highest levels of RIN1 expression in mouse are found in forebrain structures (cortex, hippocampus, amygdala, striatum, and olfactory bulb). This was determined by directed studies (Dhaka et al., 2003) and from use of available databases (GNF SymAtlas 1.0.3, symatlas.gnf.org/SymAtlas). In addition, RIN1 expression was noted in mouse testis (Dhaka et al., 2003), in mammary epithelial cells (Hu et al., 2005), and in some hematopoietic cells (Minh Thai and John Colicelli, unpublished). Limited expression studies using human tissue (Dhaka et al. [2003], GNF SymAtlas, and unpublished data) have been generally consistent with the findings reported for mouse. In many cell lines examined (HeLa, MCF7, LNCAP, JURKAT, and K562), RIN1 is found at low to moderate levels (SymAtlas). RIN1 protein can be detected by direct
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immunoblot of brain extract (60 g total protein) using appropriate antibodies (see ‘‘Reagents’’). From moderately expressing tissues (e.g., mammary epithelial cells) and cell lines (e.g., HeLa) detection of RIN1 protein requires concentration by immunoprecipitation before immunoblot analysis. Endogenous RIN1 is undetectable in the widely used HEK 293T and NIH 3T3 cell lines (Hu et al., 2005). Expression of RIN2 seems to be fairly broad in mouse tissues (SymAtlas), with some possible elevation in lung, bronchial epithelial cells, placenta, uterus, pancreatic islets, thyroid, and hematopoietic cells. In a survey of human tissues, lung, heart, and kidney showed elevated expression (Saito et al., 2002). Most human cell lines tested (HEK 293T, MCF7, LNCAP, JURKAT, K562, and PANC1) showed relatively low expression (SymAtlas). RIN3 expression in mouse is strongly elevated in hematopoietic tissues and cells including bone marrow and isolated B and T cells, as well as some cell lines derived from these sources (SymAtlas). Elevated RIN3 expression levels in human peripheral blood and spleen (Kajiho et al., 2003) are consistent with this pattern and suggest a specialized function in hematopoietic cells. Significant but lower levels of RIN3 message (mouse and human) were found in most other tissues examined. Many human cell lines (HEK 293T, MCF7, LNCAP, JURKAT, K562, PANC1, and HeLa) showed moderate levels of RIN3 expression (SymAtlas). Evidence of splice variants of all three RIN genes comes from cDNA cloning and Northern blot analysis (Han et al., 1997; Kajiho et al., 2003; Saito et al., 2002). In the case of RIN1, evidence of multiple protein products has also been seen (unpublished data). Acknowledgments The authors would like to acknowledge Ms Tay Boon Hui for technical assistance in cloning and sequencing of Takifugu rubripes cDNAs and Minh Thai for development and characterization of several unpublished antibodies. J. C. is supported by NIH grant NS046787 and DoD Breast Cancer Research Program grant W81XWH0410443.
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Christoffels, A., Koh, E. G., Chia, J. M., Brenner, S., Aparicio, S., and Venkatesh, B. (2004). Fugu genome analysis provides evidence for a whole‐genome duplication early during the evolution of ray‐finned fishes. Mol. Biol. Evol. 21, 1146–1151. Colicelli, J., Nicolette, C., Birchmeier, C., Rodgers, L., Riggs, M., and Wigler, M. (1991). Expression of three mammalian cDNAs that interfere with RAS function in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 88, 2913–2917. Dhaka, A., Costa, R. M., Hu, H., Irvin, D. K., Patel, A., Kornblum, H. I., Silva, A. J., O’Dell, T. J., and Colicelli, J. (2003). The Ras effector Rin1 modulates the formation of aversive memories. J. Neurosci. 23, 748–757. Frasca, F., Vigneri, P., Vella, V., Vigneri, R., and Wang, J. Y. (2001). Tyrosine kinase inhibitor STI571 enhances thyroid cancer cell motile response to Hepatocyte Growth Factor. Oncogene 20, 3845–3856. Han, L., and Colicelli, J. (1995). A human protein selected for interference with Ras function interacts directly with Ras and competes with Raf1. Mol. Cell. Biol. 15, 1318–1323. Han, L., Wong, D., Dhaka, A., Afar, D., White, M., Xie, W., Herschman, H., Witte, O., and Colicelli, J. (1997). Protein binding and signaling properties of RIN1 suggest a unique effector function. Proc. Natl. Acad. Sci. USA 94, 4954–4959. Hantschel, O., and Superti‐Furga, G. (2004). Regulation of the c‐Abl and Bcr‐Abl tyrosine kinases. Nat. Rev. Mol. Cell. Biol. 5, 33–44. Hernandez, S. E., Krishnaswami, M., Miller, A. L., and Koleske, A. J. (2004). How do Abl family kinases regulate cell shape and movement? Trends Cell Biol. 14, 36–44. Hu, H., Bliss, J. M., Wang, Y., and Colicelli, J. (2005). RIN1 is an ABL tyrosine kinase activator and a regulator of epithelial cell adhesion and migration. Curr. Biol. 15, 815–823. Kain, K. H., and Klemke, R. L. (2001). Inhibition of cell migration by Abl family tyrosine kinases through uncoupling of Crk‐CAS complexes. J. Biol. Chem. 276, 16185–16192. Kajiho, H., Saito, K., Tsujita, K., Kontani, K., Araki, Y., Kurosu, H., and Katada, T. (2003). RIN3: A novel Rab5 GEF interacting with amphiphysin II involved in the early endocytic pathway. J. Cell Sci. 116, 4159–4168. Rodriguez‐Viciana, P., Sabatier, C., and McCormick, F. (2004). Signaling specificity by Ras family GTPases is determined by the full spectrum of effectors they regulate. Mol. Cell. Biol. 24, 4943–4954. Saito, K., Murai, J., Kajiho, H., Kontani, K., Kurosu, H., and Katada, T. (2002). A novel binding protein composed of homophilic tetramer exhibits unique properties for the small GTPase Rab5. J. Biol. Chem. 277, 3412–3418. Segev, N. (2001). Ypt/rab gtpases: Regulators of protein trafficking. Sci. STKE 18, RE11. Szabo, K., Jekely, G., and Rorth, P. (2001). Cloning and expression of sprint, a Drosophila homologue of RIN1. Mech. Dev. 101, 259–262. Tall, G. G., Barbieri, M. A., Stahl, P. D., and Horazdovsky, B. F. (2001). Ras‐activated endocytosis is mediated by the Rab5 guanine nucleotide exchange activity of RIN1. Dev. Cell 1, 73–82. Wang, Y., Waldron, R. T., Dhaka, A., Patel, A., Riley, M. M., Rozengurt, E., and Colicelli, J. (2002). The RAS effector RIN1 directly competes with RAF and is regulated by 14‐ 3–3 proteins. Mol. Cell. Biol. 22, 916–926. Woodring, P. J., Litwack, E. D., O’Leary, D. D., Lucero, G. R., Wang, J. Y., and Hunter, T. (2002). Modulation of the F‐actin cytoskeleton by c‐Abl tyrosine kinase in cell spreading and neurite extension. J. Cell Biol. 156, 879–892. Woodring, P. J., Meisenhelder, J., Johnson, S. A., Zhou, G. L., Field, J., Shah, K., Bladt, F., Pawson, T., Niki, M., Pandolfi, P. P., Wang, J. Y., and Hunter, T. (2004). c‐Abl phosphorylates Dok1 to promote filopodia during cell spreading. J. Cell Biol. 165, 493–503.