Structurally and functionally modified forms of pp60v-src in Rous sarcoma virus-transformed cell lysates.

When analyzed from transformed cell lysates, pp60v-src, the product of the Rous sarcoma virus src gene, typically appears as a single polypeptide of 60,000 molecular weight, phosphorylated at two major sites, an amino-terminal region serine residue and carboxy-terminal region tyrosine residue. We describe here the identification of variant forms of pp60v-src present in transformed cell lysates that exhibited an altered electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels. This change in migration appeared to be the result of some alteration in the amino-terminal portion of the molecule and paralleled the appearance of extensive amino-terminal region tyrosine phosphorylation on the pp60v-src molecule. These structural modifications were further correlated with a dramatic increase in the protein kinase-specific activity of pp60v-src. The detection of these variant forms of pp60v-src depended on the prior treatment of the transformed cell cultures with vanadium ions or the inclusion in the cell disruption buffer of Mg2+ or ATP-Mg2+. The implications is that modified, highly active forms of the pp60v-src protein exist in transformed cells, but are transient and rapidly converted to stable forms, possibly by specific dephosphorylation. We suggest that amino-terminal region tyrosine phosphorylation of pp60v-src, presumably the result of autophosphorylation, serves to greatly enhance src protein enzymatic activity, but that much of the regulation of this transforming protein's function may involve a phosphotyrosyl protein phosphatase.     

Rous sarcoma virus variants that encode src proteins with an altered carboxy terminus are defective for cellular transformation.

The src gene of Rous sarcoma virus (v-src) and its cellular homolog, the c-src gene, share extensive sequence homology. The most notable differences between these genes reside in the region encoding the carboxy terminus of the src proteins. We constructed mutations within the 3' end of the v-src gene to determine the significance of this region to the transforming potential of the v-src protein, pp60v-src. The mutants CHdl300 and CHis1511 contain mutations that alter the last 23 amino acids of pp60v-src, whereas the mutant CHis1545-C contains a linker insertion that alters the last 11 amino acids of pp60v-src, and the mutant CHis1545-H contains a linker insertion that results in a 9-amino-acid insertion at position 415. Plasmids bearing each of these mutations were unable to transform chicken cells when introduced into these cells by DNA transfection. In addition, the structurally altered src proteins encoded by the mutants had much-reduced levels of tyrosine protein kinase activity in vivo, as measured by autophosphorylation and phosphorylation of the 34,000-Mr cellular protein, and in vitro, as determined by measuring the level of pp60src autophosphorylation. These data indicate that the carboxy-terminal amino acid sequences play an important role in maintaining the structure of the catalytic domain of pp60v-src. In contrast, the transfection of chicken cells with plasmid DNA containing a chimeric v-c-src gene resulted in morphological cell transformation and the synthesis of an enzymatically active hybrid protein. Therefore, the carboxy-terminal sequence alterations observed in the c-src protein do not alone serve to alter the functional activity of a hybrid v-c-src protein appreciably.     

Specific and saturable binding of pp60v-src to plasma membranes: evidence for a myristyl-src receptor

The molecular basis for membrane association of pp60v-src, the transforming protein of Rous sarcoma virus, was investigated in a cell-free system. Newly synthesized pp60v-src polypeptide, produced by in vitro translation of src mRNA, rapidly bound to plasma membranes. Binding was saturable and dependent on the presence of myristate at the amino terminus of pp60v-src. Prior treatment of membranes with heat or trypsin greatly decreased subsequent binding of pp60v-src. Membrane binding of pp60v-src was competed by a myristylated peptide containing the first 11 amino acids of the mature src sequence, but not by non-myristylated src peptide or other myristylated peptides. The specificity, saturability, and competitive nature of pp60v-src binding provide evidence for the existence of a src receptor in the plasma membrane.     

Reconstitution of the Rous sarcoma virus transforming protein pp60v-src into phospholipid vesicles.

An artificial membrane system was developed to study the molecular basis for interaction of pp60v-src, the Rous sarcoma virus transforming protein, with lipid bilayers. pp60v-src was extracted from cell membranes by detergent solubilization and reincorporated into phospholipid vesicles. Reconstituted pp60v-src retained tyrosine kinase activity and was integrally associated with the liposome through a 10-kilodalton (kDa) amino-terminal domain. The same 10-kDa domain was shown to anchor pp60v-src to the plasma membrane of transformed cells. Reconstitution experiments performed with nonmyristylated pp60v-src proteins revealed that these polypeptides did not interact with phospholipid vesicles. In contrast, myristylated, soluble pp60v-src molecules (including a highly purified pp60v-src preparation) could be reconstituted into liposomes, but their interaction with the liposomal bilayer was not mediated by the 10-kDa amino-terminal domain. When membrane proteins were included during reconstitution of purified pp60v-src, binding through the 10-kDa anchor was restored. A model is presented to accommodate the different types of interactions of pp60v-src with liposomes; the model postulates the existence of an additional membrane component that anchors the pp60v-src polypeptide to the phospholipid bilayer.     

Intracellular localization and processing of pp60v-src proteins expressed by two distinct temperature-sensitive mutants of Rous sarcoma virus.

The transforming protein of Rous sarcoma virus, pp60v-src, is known to be a tyrosine protein kinase, but the mechanism of cell transformation remains unclear. In further investigating pp60v-src structure and function, we have analyzed two temperature-sensitive (ts) Rous sarcoma virus src gene mutants, tsLA29 and tsLA32. The mutations in tsLA29 and tsLA32 map in the carboxy-terminal region and the amino-terminal half of pp60v-src, respectively, and encode mutant proteins with either temperature-labile (tsLA29) or -stable (tsLA32) kinase activities. Here we examined the intracellular processing and localization of these pp60v-src mutants and extended our characterization of transformation parameters expressed by cells infected by the Rous sarcoma virus variants. No obvious defects in functional integrity of the tsLA32 pp60v-src could yet be demonstrated, whereas the tsLA29 pp60v-src was perturbed not only in kinase activity, but also in aspects of protein processing and localization. Analysis of transformation parameters expressed by infected cells demonstrated the complete temperature lability of both mutants.     

Ecto-protein kinase activities in normal and transformed cells

The incubation of intact uninfected and Rous sarcoma virus (RSV)-transformed chicken cells (SR-RSV-A) with micromolar amounts of [gamma-32P]ATP under physiological conditions resulted in the radioactive phosphorylation of a variety of proteins. According to the experimental protocol the detectable phosphorylation was restricted to ATP utilization at the cell surface and was catalyzed by surface located protein kinase (PK). Serine- and to a lesser extent, threonine residues were phosphorylated. With respect to this enzyme the cells under investigation showed upon incubation with phosvitin the release of surface (phosvitin) kinase into the incubation medium. Based on immunochemical analysis and PK-assays using antisera from RSV-tumor bearing rabbits (TBR-serum) the pp60v-src with its associated tyrosine kinase activity was likewise detected in appreciable amounts at the outside of RSV-transformed chicken and mammalian cells. There was no cross reactivity of TBR-serum with phosvitin kinase. Phosvitin was not phosphorylated by the immunoprecipitated pp60v-src. Whereas phosphorylation catalyzed by pp60v-src was blocked with 10 to 20 microM diadenosine 5',5'-P1P4 tetraphosphate (Ap4A) the phosvitin phosphorylation was far less sensitive towards inhibition by Ap4A, similar to the cellular pp60c-src kinase activity in uninfected cells. The functional significance of the PK activities in uninfected and RSV-transformed cells observed at their surface or in cell-free form as well as the nature of their substrates remain to be established.     

Inhibition of the tyrosine kinase activity of v-src, v-fgr, and v-yes gene products by a monoclonal antibody which binds both amino and carboxy peptide fragments of pp60v-src.

A monoclonal antibody, R2D2, raised to the src gene product of Rous sarcoma virus was found to inhibit the tyrosine protein kinase activity of pp60v-src in autophosphorylation reactions and in reactions involving exogenously added substrates, such as casein and histone. R2D2 also inhibited the enzymatic activity of two related viral transforming proteins, pp70gag-fgr and pp90gag-yes. The inhibitory ability of R2D2 was dependent upon immunoglobulin concentration and could be demonstrated in both immune complexes formed directly with R2D2 and preformed immune complexes to which R2D2 was added. Binding sites in both the amino-terminal 110 amino acid residues and the carboxy-terminal 240 amino acids of pp60v-src were identified for R2D2. These results indicate that at least part of the epitope recognized by R2D2 resides within a region of the src protein which is required for protein kinase activity. The localization of the R2D2 epitope to the amino- as well as to the carboxy-terminal portions of pp60v-src, together with results of studies analyzing the relative binding efficiencies of R2D2 to the intact protein and to V-8 proteolytic fragments of pp60v-src, are consistent with the view that the R2D2 epitope is conformational in nature and that it is assembled from residues contained within both N-terminal and C-terminal regions of the molecule.     

Characterization of endogenous protein phosphorylation in isolated rat liver lysosomes

Membranes prepared from highly purified rat liver lysosomes contain endogenous protein-phosphorylation activities. The transfer of phosphate to membrane fractions from [gamma-32P]ATP was analyzed by gel electrophoresis under acidic denaturing conditions. Two phosphopeptides were detected, with molecular weights of 3,000 and 14,000. Phosphorylation of these proteins was unaffected by the addition of cAMP, cGMP, or the heat-stable inhibitor of cAMP-dependent protein kinase. No additional phosphorylation was observed when cAMP-dependent protein kinase was included in the reaction or when exogenous protein kinase substrates were added. The 14,000-dalton 32P-labeled product was formed rapidly in the presence of low concentrations (250 microM) of either Ca2+ or Mg2+. This product was labile under both acidic and alkaline conditions, suggesting that this protein contains an acyl phosphate, present presumably as a catalytic intermediate in a phosphotransferase reaction. The lower molecular weight species required a high concentration (5 mM) of Mg2+ for phosphorylation, and micromolar concentrations of Ca2+ stimulated the Mg2+-dependent activity. The addition of Ca2+ and calmodulin stimulated the phosphorylation reaction to a greater extent than with Ca2+ alone. This activity was strongly inhibited by 0.2 mM LaCl3 and to a lesser extent by 50 microM chlorpromazine or trifluoperazine. These results suggest that the 3000-dalton peptide may be phosphorylated by a Ca2+, calmodulin-dependent kinase associated with the lysosomal membrane.     

A mutation at the ATP-binding site of pp60v-src abolishes kinase activity, transformation, and tumorigenicity.

We constructed a mutant, called RSV-SF2, at the ATP-binding site of pp60v-src. In this mutant, lysine-295 is replaced with methionine. SF2 pp60v-src was found to have a half-life similar to that of wild-type pp60v-src and was localized in the membranous fraction of the cell. Rat cells expressing SF2 pp60v-src were morphologically untransformed and do not form tumors. The SF2 pp60v-src isolated from these cells lacked kinase activity with either specific immunoglobulin or other substrates, and expression of SF2 pp60v-src failed to cause an increase of total phosphotyrosine in the proteins of infected cells. Wild-type pp60v-src was phosphorylated on serine and tyrosine in infected cells, and the analogous phosphorylations could also be carried out in vitro. Phosphorylation of serine was catalyzed by a cyclic AMP-dependent protein kinase, and phosphorylation of tyrosine was perhaps catalyzed by pp60v-src itself. By contrast, SF2 pp60v-src could not be phosphorylated on serine or tyrosine either in infected cells or in vitro. These findings strengthen the belief that the phosphotransferase activity of pp60v-src is required for neoplastic transformation by the protein and suggest that the binding of ATP to pp60v-src elicits an allosteric change required for phosphorylation of serine in the protein.     

5''-flanking sequences that inhibit in vitro transcription of a xenopus laevis tRNA gene

We determined the nucleotide sequences of all coding regions and a significant part of the flanking regions of the chicken c-src gene, which is a cellular homolog of the v-src gene of Rous sarcoma virus. The c-src gene consists of 12 exons; the boundaries of the exons were determined by assuming that the amino acid sequence of its product, pp60c-src, is basically the same as that of pp60v-src. The deduced amino acid sequence of pp60c-src was very similar to that of pp60v-src, but the last 19 carboxy-terminal amino acids of pp60c-src were replaced by a new set of 12 amino acids of pp60v-src. The sequence encoding the carboxy-terminal sequence of pp60v-src was found 900 bp downstream from the termination codon of the c-src gene. We suggest that the c-src sequence was captured by a virus through recombination at both sides of the c-src gene, and that the recombinations occurred at the level of proviral DNA.     

Highly specific antibody to Rous sarcoma virus src gene product recognizes a novel population of pp60v-src and pp60c-src molecules

Antiserum to the Rous sarcoma virus (RSV)-transforming protein, pp60v-src, was produced in rabbits immunized with p60 expressed in Escherichia coli. alpha p60 serum immunoprecipitated quantitatively more pp60v-src than did tumor-bearing rabbit (TBR) sera. When RSV-transformed cell lysates were preadsorbed with TBR serum, the remaining lysate contained additional pp60v-src, which was recognized only by reimmunoprecipitation with alpha p60 serum and not by TBR serum. In subcellular fractions of RSV-infected chicken embryo fibroblasts (RSV-CEFs) and field vole cells probed with TBR serum, the majority of the pp60v-src was associated with the plasma membrane-enriched P100 fraction. However, alpha p60 serum revealed equal distribution of pp60v-src and its kinase activity between the P1 (nuclear) and P100 fractions. The same results were obtained for pp60c-src in uninfected CEFs. On discontinuous sucrose gradients nearly 50% of the P1-pp60v-src sedimented with nuclei, in fractions where no plasma membrane was detected. Indirect immunofluorescence microscopy of RSV-CEFs with alpha p60 serum revealed a distinct pattern of perinuclear fluorescence, in addition to staining at the cell periphery. Thus the use of a highly specific antibody reveals that enzymatically active pp60v-src and pp60c-src molecules are present in other intracellular structures, probably juxtareticular nuclear membranes, in addition to the plasma membrane in normal, uninfected, and wild-type RSV-infected cells.     

Vmax. activation of pp60c-src tyrosine kinase from neuroblastoma neuro-2A.

A kinetic analysis of the tyrosine-specific protein kinase of pp60c-src from the C1300 mouse neuroblastoma cell line Neuro-2A and pp60c-src expressed in fibroblasts was carried out to determine the nature of the increased specific activity of the neuroblastoma enzyme. In immune-complex kinase assays with ATP-Mn2+ and the tyrosine-containing peptide angiotensin I as phosphoacceptor substrate, pp60c-src from the neuroblastoma cell line was characterized by a maximum velocity (Vmax.) that was 7-15-fold greater than the Vmax. of pp60c-src from fibroblasts. The neuroblastoma enzyme exhibited Km values for ATP (16 +/- 3 microM) and angiotensin I (6.8 +/- 2.6 mM) that were similar to Km values for ATP (25 +/- 3 microM) and angiotensin I (6.5 +/- 1.7 mM) of pp60c-src from fibroblasts. pp60v-src expressed in Rous-sarcoma-virus-transformed cells exhibited an ATP Km value (25 +/- 4 microM) and an angiotensin I Km value (6.6 +/- 0.5 mM) that approximated the values determined for pp60c-src in neuroblastoma cells and fibroblasts. These results indicate that the pp60c-src kinase from neuroblastoma cells has a higher turnover number than pp60c-src kinase from fibroblasts, and that the neural form of the enzyme would be expected to exhibit increased catalytic activity at the saturating concentrations of ATP that are found intracellularly.     

Phosphatidylinositol kinase activities in normal and Rous sarcoma virus-transformed cells.

The phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), and diacylglycerol kinase activities in the plasma membrane-rich fraction of chicken embryo fibroblasts infected with a temperature-sensitive mutant of Rous sarcoma virus increased when the cells were shifted from the nonpermissive temperature, 41 degrees C, to the permissive temperature, 35 degrees C. Temperature shift from 35 to 41 degrees C decreased the lipid kinase activities in the membrane vesicles. These changes accompanied the changes observed in pp60v-src protein kinase activity. Thermal inactivation at 41 degrees C did not appreciably reduce PI and PIP kinase activities in membrane vesicles prepared from uninfected or Rous sarcoma virus-transformed cells, whereas pp60v-src protein kinase activity in the membrane vesicles was rapidly inactivated under the same conditions. These data suggest that pp60v-src may indirectly enhance PI and PIP phosphorylation but not directly contribute to this pathway.     

Restriction of the in vitro and in vivo tyrosine protein kinase activities of pp60c-src relative to pp60v-src.

The tyrosine protein kinase activities of pp60c-src and pp60v-src were compared. The activities were qualitatively similar in vitro when the src proteins were bound in an immune complex with monoclonal antibody; both proteins utilized either ATP or GTP as phosphate donors, preferred Mn2+ to Mg2+, and had similar exogenous substrate specificities. The specific activity of pp60c-src was about 10-fold lower than that of pp60v-src for exogenous substrate phosphorylation but was only 1.1- to 2-fold lower than that of pp60v-src for autophosphorylation. Six glycolytic enzymes, including three not previously identified as substrates for pp60src phosphorylation, were phosphorylated by both pp60c-src and pp60v-src. Levels of pp60c-src fourfold higher than the amount of pp60v-src in src-plasmid-transformed cells did not detectably alter the level of phosphotyrosine in cellular proteins, but increasing the expression of pp60c-src another twofold (which induces cells to form foci in monolayer culture (P.J. Johnson, P.M. Coussens, A.V. Danko, and D. Shalloway, Mol. Cell. Biol. 5:1073-1083, 1985) resulted in a threefold increase in the level of cellular protein phosphotyrosine. Immunoprecipitation and analysis of the alkali-stable phosphoproteins by two-dimensional electrophoresis showed that, in contrast to pp60v-src-transformed cells, pp36 and enolase are only weakly phosphorylated in these high-level pp60c-src overexpresser cells. Even allowing for the in vitro differences in specific activities of phosphorylation, these results suggest that the pp60c-src tyrosine protein phosphorylating activity may be restricted relative to that of pp60v-src by additional in vivo mechanisms.     

Altered sites of tyrosine phosphorylation in pp60c-src associated with polyomavirus middle tumor antigen.

We characterized the tyrosine phosphorylation sites of free pp60c-src and of pp60c-src associated with the polyomavirus middle tumor antigen (mT) in transformed avian and rodent cells. The sites of tyrosine phosphorylation in the two populations of pp60c-src were different, both in vitro and in vivo. Free pp60c-src was phosphorylated in vitro at a single site, tyrosine 416. pp60c-src associated with mT was phosphorylated in vitro on tyrosine 416 and on one or more additional tyrosine residues located in the amino-terminal region of the molecule. Free pp60c-src in polyomavirus mT-transformed cells was phosphorylated in vivo on tyrosine 527. In contrast, pp60c-src associated with mT was phosphorylated in vivo on tyrosine 416 and not detectably on tyrosine 527. Thus, the in vivo phosphorylation sites of pp60c-src associated with mT in transformed cells are identical to those of pp60v-src, the Rous sarcoma virus transforming protein. The results suggest that altered phosphorylation of pp60c-src associated with mT may play a role in the enhancement of the pp60c-src protein kinase activity and in cell transformation by polyomavirus.     

A mutation in v-src that removes a single conserved residue in the SH-2 domain of pp60v-src restricts transformation in a host-dependent manner.

The v-src oncogene of Rous sarcoma virus (RSV) is able to transform both avian and mammalian cells, but the mutant allele v-src-L displays a host range dependence for transformation, transforming chicken but not rat cells with wild-type efficiency. This host range restriction can be detected by measuring growth in low serum, saturation density, and anchorage independent growth. In addition, rat cells expressing v-src-L do not form tumors in syngeneic rats or nude mice, but RSV carrying the mutant allele causes tumors in chicks, although at a reduced efficiency and with increased latency. To determine the lesion responsible for this phenotype, we sequenced the entire v-src gene from the parental B77 strain of RSV, as well as the mutant allele. v-src-L is missing 3 nucleotides present in the wild-type parent, RSV B31, eliminating Phe-172, an invariant residue in a conserved region of src-related proteins known as SH-2. The kinase activity of pp60v-src-L was indistinguishable from that of the wild type in chicken cells but was significantly reduced in rat cells as assayed by an in vitro immune complex assay; in vivo phosphorylation of one specific substrate, p36 (calpactin I heavy chain); and total phosphotyrosine-containing proteins. In addition, the pattern of phosphotyrosine-containing proteins in rat cells was qualitatively different when cells containing pp60v-src-L were compared with cells with wild-type pp60v-src, even though both pp60v-src proteins were membrane associated. The data are consistent with a role for the SH-2 region in substrate specificity.     

Inter- and intramolecular interactions of highly purified Rous sarcoma virus-transforming protein, pp60v-src

We have purified intact pp60v-src, the product of the Rous sarcoma virus src gene, over 2400-fold, based on the phosphorylation of tumor-bearing rabbit IgG. The purification procedure involved detergent extraction of the particulate fraction of the cells and sequential chromatography on hydroxylapatite, butyl agarose, DEAE-Sephacel, ADP-agarose, and Sephacryl S-200. Analysis of the preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single silver-stained band with an apparent molecular weight of 60,000. Our results show that the activities of this preparation were qualitatively similar to those described previously for partially purified pp60v-src. Upon analysis by two-dimensional gel electrophoresis, the purified pp60v-src yielded one major species which migrated to the same position as the least acidic of the three major species detectable in cellular lysates, suggesting that the pp60v-src had been dephosphorylated during the purification procedure. We found that pp60v-src was very prone to aggregation; to maintain it as a monomer both Nonidet P-40 and KCl were required. Under conditions which maintained pp60v-src as a monomer, the rate of autophosphorylation was independent of its concentration and thus proceeded via an intramolecular process. Preincubation of pp60v-src with ATP or GTP as well as nonphosphorylating analogs of ATP or GTP preserved its phosphorylating activity toward alpha-casein whereas its activity was reduced 80% upon preincubation in the absence of nucleotides. We suggest that protection with nucleotides rather than autophosphorylation accounts for the apparent increase in the activity of pp60v-src after incubation of the enzyme with ATP.     

Characterization of the Rous sarcoma virus transforming gene product

This report describes quantitative results of in vitro phosphorylation of the Rous sarcoma virus transforming gene product, pp60src, using ATP or GTP as phosphate donors. The Km values for the phosphorylation of pp60src by ATP and GTP were similar (10-36 and 25-36 microM, respectively) and the Vmax values were different (5-7 and 1.5-1.7 nmol min-1 mg-1 of pp60src, respectively). The radiolabeling of pp60src by [gamma-32P] ATP was inhibited by ADP and dATP at 20-fold higher concentrations by 75 and 83%, respectively. Other nucleotides served as weaker inhibitors under the same conditions. The radiolabeling of pp60src by [gamma-32P]GTP had lower specificity for this nucleotide, since ATP, dATP, ADP, dGTP, GDP, and TTP had at least a 50% inhibitory effect. The phosphorylated products of approximate Mr = 60,000 that were produced with ATP or GTP were shown to be the same protein molecule since they both could be immunoprecipitated with antibody raised against p60src produced by bacterial recombinants. Structural analysis revealed that the use of GTP resulted in phosphorylation of a tyrosine residue on the COOH-terminal region of pp60src, apparently the same site which contains the tyrosine phosphorylated in infected cells. In contrast, the use of ATP resulted in phosphorylation of several additional tyrosine residues on the NH2-terminal region of the molecule. In thermolability studies, the t1/2 values for the phosphorylation of pp66src in preparations from wild type virus-infected chicken cells were 5.1 min for both ATP and GTP, whereas the t1/2 values for the phosphorylation of pp60src in preparations from temperature-sensitive transformation mutant-infected cells were 1.1 min for both phosphate donors. Similar observations were found with alpha-casein as substrate.     

Genetic analysis of p60v-src domains involved in the induction of different cell transformation parameters.

The expression of p60v-src in chicken cells infected with Rous sarcoma virus causes stimulation of cell proliferation, morphological alteration, and anchorage independence. PA101 and PA104 are temperature-sensitive variants encoding mutant p60v-src proteins that are partially defective in the induction of these transformation parameters. To define the structural basis for the transformation defectiveness of the p60v-src mutants, the v-src genes of PA101 and PA104 were molecularly cloned and analyzed. Amino- and carboxy-terminal coding regions of the cloned mutant genes were exchanged with the corresponding regions of cloned wild-type v-src and chicken c-src genes, reconstructed into viral DNA, and expressed in infected cells maintained at various temperatures. This analysis revealed that lesions within the tyrosine kinase domains of the two mutant proteins confer temperature sensitivity on all three transformation functions of p60v-src. An amino-terminal region of the PA101 mutant protein, which coincides with the proposed modulatory domain and appears to interact with the kinase domain, affects morphological alteration in a temperature-independent manner. Our results suggest that the function of the kinase domain is essential to all three parameters examined, whereas the amino-terminal domain is important in determining cell morphology.