Polyomavirus middle T-antigen NPTY mutants.

A polyomavirus middle T-antigen (MTAg) mutant containing a substitution of Leu for Pro at amino acid 248 has previously been described as completely transformation defective (B. J. Druker, L. Ling, B. Cohen, T. M. Roberts, and B. S. Schaffhausen, J. Virol. 64:4454-4461, 1990). This mutant had no alterations in associated proteins or associated kinase activities compared with wild-type MTAg. Pro-248 lies in a tetrameric sequence, NPTY, which is reminiscent of the so-called NPXY sequence in the low-density-lipoprotein receptor. In the low-density-lipoprotein receptor, mutations in the NPXY motif but not in the surrounding amino acids abolish receptor function, apparently by decreasing receptor internalization (W. Chen, J. L. Goldstein, and M. S. Brown, J. Biol. Chem. 265:3116-3123, 1990). To determine whether this sequence represents a functional motif in MTAg as well, a series of single amino acid substitutions was constructed in this region of MTAg. All of the mutations of N, P, T, or Y, including the relatively conservative substitution of Ser for Thr at amino acid 249, resulted in a transformation-defective MTAg, whereas mutations outside of this sequence allowed mutants to retain near-wild-type transformation capabilities. Transformation-defective mutants with mutations in the NPTY region behaved similarly to the mutant with the original Pro-248-to-Leu-248 mutation when assayed for associated proteins and activities in vitro; that is, they retained a full complement of wild-type activities and associated proteins. Further, insertion of the tetrameric sequence NPTY downstream of the mutated motif restored transforming abilities to these mutants. Thus, the tetrameric sequence NPTY in MTAg appears to represent a well-defined functional motif of MTAg.     

Transformation-defective mutants of polyomavirus middle T antigen associate with phosphatidylinositol 3-kinase (PI 3-kinase) but are unable to maintain wild-type levels of PI 3-kinase products in intact cells.

Middle T antigen (MT) of polyomavirus causes transformation by associating with a number of cellular proteins. The association with and activation of two such proteins, phosphatidylinositol 3-kinase (PI 3-kinase) and pp60c-src, appears to be necessary for transformation by MT. The tyrosine kinase activity of MT-associated pp60c-src is significantly increased when assayed in vitro, and levels of phosphotyrosine-containing proteins are elevated in vivo. Similarly, levels of the PI 3-kinase products phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2] and phosphatiylinositol-3,4,5-trisphosphate [PI(3,4,5)P3] are constitutively elevated in MT-transformed cells. However, the formation of a complete MT/cellular protein complex and the activation of tyrosine kinase are not sufficient to cause transformation, since the transformation-defective mutants 248m and dl1015 associate with all wild-type MT-associated proteins, including PI 3-kinase and pp60c-src, and neither mutant appears to be defective in MT-associated tyrosine kinase activity. Studies presented here compared (i) the amount of PI 3-kinase activity associated with the MT complex and (ii) levels of [3H]inositol incorporation into PI 3-kinase products in cells expressing mutant or wild-type MT. The results show that dl1015 is defective in both assays, whereas 248m is defective only for incorporation of [3H]inositol into PI(3,4,5)P2 and PI(3,4)P3. These findings identify a biochemical defect in the 248m mutant and corroborate previous results correlating transformation and elevated levels of PI 3-kinase products in vivo. In addition, they indicate that PI 3-kinase product levels are affected by factors other than simply the amount of PI 3-kinase activity associated with the MT complex.     

Structural and functional modification of pp60c-src associated with polyoma middle tumor antigen from infected or transformed cells.

The polyoma middle tumor antigen (MTAg) associates with the src proto-oncogene product pp60c-src in infected or transformed rodent cells. The tyrosine protein kinase activity of pp60c-src, as measured by in vitro phosphorylation of pp60c-src itself or the exogenous substrate enolase, was increased 10- to 20-fold in cells transformed or infected with transformation-competent polyoma virus compared with controls. pp60c-src associated with MTAg and precipitated with polyoma antitumor serum had a novel site(s) of in vitro tyrosine phosphorylation within its amino-terminal domain. These observations suggest that association of MTAg with pp60c-src alters the accessibility of pp60c-src tyrosine residues for phosphorylation in vitro and increases pp60c-src protein kinase activity. Several transformation-defective mutants of MTAg did not cause amino-terminal tyrosine phosphorylation of pp60c-src in vitro or enhance its protein kinase activity, suggesting that these properties correlate with the transforming ability of MTAg. However, one transformation-defective MTAg mutant, dl1015, did cause amino-terminal tyrosine phosphorylation of pp60c-src in vitro and did enhance its protein kinase activity. This suggests that properties of MTAg, in addition to modifying the structure and function of pp60c-src, may be important for transformation.     

Common elements in growth factor stimulation and oncogenic transformation: 85 kd phosphoprotein and phosphatidylinositol kinase activity

The phosphorylation of proteins on tyrosine in vivo and in vitro was examined in 3T3 cells stimulated by platelet-derived growth factor (PDGF) and transformed by polyoma middle T antigen (MTAg) by using an antibody directed against phosphotyrosine (P-tyr). Two common events were observed upon PDGF stimulation or MTAg transformation of cells: the appearance in the immunoprecipitates of an 85 kd phosphoprotein, and increased phosphatidylinositol (PI) kinase activity. In PDGF-stimulated cells, the 85 kd phosphoprotein and PI kinase activity appeared rapidly, within 1 min of growth factor addition. The PI kinase activity and 85 kd phosphorylation were also increased in anti-P-tyr immunoprecipitates from cells transformed by v-fms and v-sis, but not by SV40 T antigen. The presence of the tyrosine-phosphorylated 85 kd protein correlated with PI kinase activity during several purification steps. These results suggest that the 85 kd phosphoprotein, a putative PI kinase, is a substrate for both the PDGF receptor and MTAg/pp60c-src tyrosine kinase activities.     

Phosphoinositide kinase activity and transformation

We have used the DNA tumor virus polyoma as a model system to examine whether the phosphatidylinositol (PI) turnover pathway is a critical target for transforming gene products. Polyoma-infected cells show elevated levels of polyphosphoinositides and polyphosphoinositols, and a PI kinase activity is associated with middle T antigen, a transforming gene product of polyoma virus. In anti-T immunoprecipitates from polyoma-infected or -transformed cells, comparisons of wild-type and polyoma mutants defective for transformation show a strong correlation between middle T-associated PI kinase activity and transforming ability. Middle T has previously been found to associate at the plasma membrane with pp60 c-src and to activate it as a tyrosine kinase. c-src itself does not appear to phosphorylate PI; however, the middle T/pp60 c-src tyrosine kinase activity may be important for activation of PI kinase. Ammonium orthovanadate, a tyrosine phosphatase inhibitor, elevates the middle T/pp60 c-src-associated PI kinase activity. We propose that middle T/pp60 c-src activates a PI kinase and modulates PI turnover in vivo by tyrosine phosphorylation.     

Deletion or substitution within the alpha platelet-derived growth factor receptor kinase insert domain: effects on functional coupling with intracellular signaling pathways.

The tyrosine kinase domains of the platelet-derived growth factor (PDGF) and colony-stimulating factor-1 (CSF-1)/c-fms receptors are interrupted by kinase inserts (ki) which vary in length and amino acid sequence. To define the role of the ki in the human alpha PDGF receptor (alpha PDGFR), we generated deletion mutants, designated alpha R delta ki-1 and alpha R delta ki-2, which lacked 80 (710 to 789) and 95 (695 to 789) amino acids of the 104-amino-acid ki region, respectively. Their functional characteristics were compared with those of the wild-type alpha PDGFR following introduction into a naive hematopoietic cell line, 32D. Biochemical responses, including PDGF-stimulated PDGFR tyrosine phosphorylation, phosphatidylinositol (PI) turnover, and receptor-associated PI-3 kinase activity, were differentially impaired by the deletions. Despite a lack of any detectable receptor-associated PI-3 kinase activity, 32D cells expressing alpha R delta ki-1 showed only partially impaired chemotactic and mitogenic responses and were capable of sustained proliferation in vitro and in vivo under conditions of autocrine stimulation by the c-sis product. 32D transfectants expressing the larger ki deletion (alpha R delta ki-2) showed markedly decreased or abolished biochemical and biological responses. However, insertion of the highly unrelated smaller c-fms (685 to 750) ki domain into alpha R delta ki-2 restored each of these activities to wild-type alpha PDGFR levels. Since the CSF-1R does not normally induce PI turnover, the ability of the c-fms ki domain to reconstitute PI turnover in the alpha R delta ki-2 transfectant provides evidence that the ki domain of the alpha PDGFR does not directly couple with this pathway. Taken together, all od these bindings imply that their ki domains have evolved to play very similar roles in the known signaling functions PDGF and CSF-1 receptors.     

A short sequence in the p60src N terminus is required for p60src myristylation and membrane association and for cell transformation.

We have constructed mutants by using linker insertion followed by deletion in the region of cloned Rous sarcoma virus DNA coding for the N-terminal 9 kilodaltons of the src protein. Previous work implicated this region in the membrane association of the protein. The mutations had little effect on src tyrosine kinase activity. Substitution of a tri- or tetrapeptide for amino acids 15 to 27, 15 to 49, or 15 to 81 had little effect on the in vitro transforming capacity of the virus. Like wild-type p60src, the src proteins of these mutants associated with plasma membranes and were labeled with [3H]myristic acid. In contrast, a mutant whose src protein had the dipeptide Asp-Leu substituted for amino acids 2 to 81 and a mutant with the tripeptide Asp-Leu-Gly substituted for amino acids 2 to 15 were transformation defective, and the mutant proteins did not associate with membranes and were not labeled with [3H]myristic acid. These results suggest that amino acids 2 to 15 serve as an attachment site for myristic acid and as a membrane anchor. Since deletions including this region prevent transformation, and since tyrosine kinase activity is not diminished by the deletions, these results imply that target recognition is impaired by mutations altering the very N terminus, perhaps through their effect on membrane association.     

Analysis of middle tumor antigen and pp60c-src interactions in polyomavirus-transformed rat cells.

The relative abundance of pp60c-src molecules associated with polyomavirus (Py) middle tumor antigen (MTAg) and the relative abundance of MTAg associated with pp60c-src in a variety of Py-transformed rat cells was determined by quantitative immunoblot analyses which detect pp60c-src or Py MTAg. The results demonstrate that approximately 5 to 10% of the total immunoprecipitable pp60c-src molecules in Py-transformed rat cells are stably associated with MTAg and have elevated protein kinase activities. In these same cells, it was found that approximately 10 to 15% of the detectable MTAg molecules are stably associated with pp60c-src. Other results presented in this report demonstrate that approximately 50 to 75% of the total MTAg-associated cellular tyrosine kinase activity potentially represents the enzymatic activity of pp60c-src, while the remaining 25 to 50% represents the activity of other cellular tyrosine kinases. Our results also show that most pp60c-src molecules associated with Py MTAg do not possess electrophoretic mobilities that are altered from those of pp60c-src molecules not associated with MTAg or pp60c-src molecules obtained from normal rodent cells.     

Transformation-defective polyoma middle T antigen mutants defective in PLCgamma, PI-3, or src kinase activation enhance ERK2 activation and promote retinoic acid-induced, cell differentiation like wild-type middle T

In HL-60 human myeloblastic leukemia cells, retinoic acid is known to cause cFMS, RAF, MEK, and ERK2 dependent myeloid cell differentiation and G0 arrest associated with RB tumor suppressor protein hypophosphorylation, implicating receptor tyrosine kinase signal transduction in propelling these retinoic acid-induced cellular effects. Furthermore, ectopic expression of polyoma middle T antigen, which activates similar early signal transduction molecules as PDGF class receptors such as cFMS, accelerates these retinoic acid-induced effects. To determine if this depends on middle T's ability to activate PLCgamma, PI-3 kinase, and src-like kinases, stable transfectants of HL-60 cells expressing either the polyoma middle T dl23 mutant, which is defective for PLCgamma and PI-3 kinase activation, or the Delta205 mutant, which in addition has greatly attenuated src-like kinase activation ability, were created and compared to wild-type middle T-transfected HL-60. The transgenes were under control of the retinoic acid (or 1, 25-dihydroxy vitamin D3) inducible Moloney murine leukemia virus LTRs. Expression of the dl23 or Delta205 mutant accelerated retinoic acid-induced cell differentiation. The effects of the mutants were comparable to those of the wild-type middle T. Likewise, retinoic acid-induced G0 arrest of mutant transfected cells and wild-type middle T transfected cells was similar. The same was true for 1, 25-dihydroxy vitamin D3-induced monocytic differentiation as for retinoic acid-induced myeloid differentiation. The mutants did not cause the same slight shortening of the cell cycle as wild-type middle T. Both the mutants and the wild-type middle T caused a similar increase in the cellular basal level of activated ERK2 MAPK. Since retinoic acid increases ERK2 activation, which is necessary for differentiation, the data suggest that mutant and wild-type middle T enhanced the retinoic acid effects by increasing basal levels of ERK2 activation. Consistent with this, the polyoma-induced foreshortening of the time for differentiation coincided with the time for retinoic acid to significantly increase ERK2 activation. As in wild-type HL-60, retinoic acid induced the early down-regulation of RXRalpha in mutant transfectants similar to wild-type middle T transfectants, consistent with no loss or gain of relevant functions due to the mutations. In contrast, vitamin D3 did not down-regulate RXRalpha in HL-60 or transfectants. Polyoma middle T and these transformation-defective mutants thus enhanced ERK2 activation to have an early effect in promoting retinoic acid-induced differentiation without a strong dependence on activating PLCgamma, PI-3 kinase, or src-like kinase.     

Biological function of PDGF-induced PI-3 kinase activity: its role in alpha PDGF receptor-mediated mitogenic signaling

The tyrosine phosphorylation sites in the human alpha PDGF receptor (alpha PDGFR) required for association with PI-3 kinase have been identified as tyrosines 731 and 742. Mutation of either tyrosine substantially reduced PDGF-induced PI-3 kinase activity but did not impair the receptor-mediated mitogenic response. We sought to determine whether PDGF-induced PI-3 kinase activity could be further ablated so as to exclude a low threshold requirement for PDGFR signal transduction. Thus, we mutated both tyrosine 731 and 742 and expressed the double mutant (Y731F/Y742F) in 32D hematopoietic cells. In such transfectants, PDGF induced no detectable receptor-associated or anti-P- Tyr recoverable PI-3 kinase activity. Under the same conditions, neither mobility shift of raf-1 nor tyrosine phosphorylation of either PLC gamma or MAP kinase was impaired. 32D transfectants expressing the double mutant showed wild-type alpha PDGFR levels of mitogenic and chemotactic responses to PDGF. To examine the effect of the double mutation in cells that normally respond to PDGF, we generated chimeras in which the cytoplasmic domains of wild-type alpha PDGFR, Y731F, and Y731F/Y742F were linked to the extracellular domain of colony- stimulating factor-1 (CSF-1) receptor (fms). After introduction of the chimeric receptors into mouse NIH/3T3 fibroblasts, the ability of CSF-1 to stimulate growth of these transfectants was examined. Our data show that all these chimeric receptors exhibited similar abilities to mediate CSF-1-stimulated cell growth. These findings lead us to conclude that PDGF-induced PI-3 kinase activity is not required for PDGF-stimulated mitogenic pathway in both NIH/3T3 fibroblasts and 32D hematopoietic cells.     

Activation of the JNK pathway is essential for transformation by the Met oncogene.

The Met/Hepatocyte Growth Factor (HGF) receptor tyrosine kinase is oncogenically activated through a rearrangement that creates a hybrid gene Tpr-Met. The resultant chimeric p65(Tpr-Met) protein is constitutively phosphorylated on tyrosine residues in vivo and associates with a number of SH2-containing signaling molecules including the p85 subunit of PI-3 kinase and the Grb2 adaptor protein, which couples receptor tyrosine kinases to the Ras signaling pathway. Mutation of the binding site for Grb2 impairs the ability of Tpr-Met oncoprotein to transform fibroblasts, suggesting that the activation of the Ras/MAP kinase signaling pathway through Grb2 may be essential for cellular transformation. To test this hypothesis dominant-negative mutants of Grb2 with deletions of the SH3 domains were introduced into Tpr-Met transformed fibroblasts. Cells overexpressing the mutants were found to be morphologically reverted and exhibited reduced growth in soft agar. Surprisingly, the Grb2 mutants blocked activation of the JNK/SAPK but not MAP kinase activity induced by the Tpr-Met oncoprotein. Additionally, cells expressing dominant-negative Grb2 mutants had reduced PI-3-kinase activity and dominant-negative mutants of Rac1 blocked both Tpr-Met-induced transformation and activation of JNK. These experiments reveal a novel link between Met and the JNK pathway, which is essential for transformation by this oncogene.     

Aromatic amino acid biosynthesis: gene-enzyme relationships in Bacillus subtilis

Single step mutants of Bacillus subtilis which required either one or all of the aromatic amino acids for growth were isolated. The relevant gene defect was determined for each mutant by enzyme assays in vitro. A mutant deficient in each enzyme step of aromatic amino acid biosynthesis was found with the exceptions of the shikimate kinase and the phenylalanine and tyrosine transaminases. Representative mutants carrying the defective genes were mapped by deoxyribonucleic acid mediated transformation by reference to the aromatic amino acid gene (aro) cluster and, alternately, to any of the other unlinked aro genes. The genes coding for dehydroquinate synthetase, 3-enol pyruvylshikimate 5-phosphate synthetase, one form of chorismate mutase, and prephenate dehydrogenase are linked to the aro cluster. Except for the previously identified linkage between the genes of 3-deoxy-d-arabino heptulosonic acid 7-phosphate synthetase and one species of chorismate mutase, the other genes involved in this pathway are neither linked to the aro cluster nor to each other.     

Rous sarcoma virus mutant dlPA105 induces different transformed phenotypes in quail embryonic fibroblasts and neuroretina cells.

dlPA105 is a spontaneous variant of Rous sarcoma virus, subgroup E, which carries a deletion in the N-terminal portion of the v-src gene coding sequence. This virus was isolated on the basis of its ability to induce proliferation of quiescent quail neuroretina cells. The altered v-src gene encodes a phosphoprotein of 45,000 daltons which possesses tyrosine kinase activity. DNA sequencing of the mutant v-src gene has shown that deletion extends from amino acid 33 to 126 of wild-type p60v-src. We investigated the tumorigenic and transforming properties of this mutant virus. dlPA105 induced fibrosarcomas in quails with an incidence identical to that induced by wild-type virus. Quail neuroretina cells infected with the mutant virus were morphologically transformed and formed colonies in soft agar. In contrast, dlPA105 induced only limited morphological alterations in quail fibroblasts and was defective in promoting anchorage-independent growth of these cells. Synthesis and tyrosine kinase activity of the mutant p45v-src were similar in both cell types. These data indicate that the portion of the v-src protein deleted in p45v-src is dispensable for the mitogenic and tumorigenic properties of wild-type p60v-src, whereas it is required for in vitro transformation of fibroblasts. The ability of dlPA105 to induce different transformation phenotypes in quail fibroblasts and quail neuroretina cells is a property unique to this Rous sarcoma virus mutant and provides evidence for the existence of cell-type-specific response to v-src proteins.     

Anti-oncogenic activity of signalling-defective epidermal growth factor receptor mutants.

Overexpression and autocrine activation of the epidermal growth factor receptor (EGF-R) cause transformation of cultured cells and correlate with tumor progression in cancer patients. Dimerization and transphosphorylation are crucial events in the process by which receptors with tyrosine kinase activity generate normal and transforming cellular signals. Interruption of this process by inactive receptor mutants offers the potential to inhibit ligand-induced cellular responses. Using recombinant retroviruses, we have examined the effects of signalling-incompetent EGF-R mutants on the growth-promoting and transforming potential of ligand-activated, overexpressed wild-type EGF-R and the v-erbB oncogene product. Expression of a soluble extracellular EGF-R domain had little if any effect on the growth and transformation of NIH 3T3 cells by either tyrosine kinase. However, both a kinase-negative EGF-R point mutant (HERK721A) and an EGF-R lacking 533 C-terminal amino acids efficiently inhibited wild-type EGF-R-mediated, de novo DNA synthesis and cell transformation in a dose-dependent manner. Furthermore, coexpression with the v-erbBES4 oncogene product in NIH 3T3 cells resulted in transphosphorylation of the HERK721A mutant receptor and reduced soft-agar colony growth but had no effect in a focus formation assay. These results demonstrate that signalling-defective receptor tyrosine kinase mutants differentially interfere with oncogenic signals generated by either overexpressed EGF-R or the retroviral v-erbBES4 oncogene product.     

Mechanisms of transformation by polyoma virus middle T antigen

This review addresses a fundamental question of polyoma virus biology: What is the molecular mechanism by which the polyoma virus middle T antigen (MTAg) transforms cells in culture? Since MTAg has no known intrinsic biochemical activity, it is believed to act by modulating the properties of the host cell's proteins (see review by Courtneidge [26]). Experiments to date have largely focused on the interaction of MTAg with the cellular tyrosine kinase, pp60c-src. However, recent data from a number of laboratories have demonstrated the importance of other MTAg-associating cellular proteins in MTAg-mediated transformation, including pp62c-yes and a phosphatidylinositol kinase. In this review, we will summarize what is presently known about the proteins interacting with MTAg. The extent to which the currently known details of the biochemistry of MTAg and its associated proteins can explain the transforming properties of the various mutant alleles of MTAg will be assessed.     

GTPase-activating protein and phosphatidylinositol 3-kinase bind to distinct regions of the platelet-derived growth factor receptor beta subunit.

In response to binding of platelet-derived growth factor (PDGF), the PDGF receptor (PDGFR) beta subunit is phosphorylated on tyrosine residues and associates with numerous signal transduction enzymes, including the GTPase-activating protein of ras (GAP) and phosphatidylinositol 3-kinase (PI3K). Previous studies have shown that association of PI3K requires phosphorylation of tyrosine 751 (Y751) in the kinase insert and that this region of receptor forms at least a portion of the binding site for PI3K. In this study, the in vitro binding of GAP to the PDGFR was investigated. Like PI3K, GAP associates only with receptors that have been permitted to autophosphorylate, and GAP itself does not require tyrosine phosphate in order to stably associate with the phosphorylated PDGFR. To define which tyrosine residues are required for GAP binding, a panel of PDGFR phosphorylation site mutants was tested. Mutation of Y771 reduced the amount of GAP that associates to an undetectable level. In contrast, the F771 (phenylalanine at 771) mutant bound wild-type levels of PI3K, whereas the F740 and F751 mutants bound 3 and 23%, respectively, of the wild-type levels of PI3K but wild-type levels of GAP. The F740/F751 double mutant associated with wild-type levels of GAP, but no detectable PI3K activity, while the F740/F751/F771 triple mutant could not bind either GAP or PI3K. The in vitro and in vivo associations of GAP and PI3K activity to these PDGFR mutants were indistinguishable. The distinct tyrosine residue requirements suggest that GAP and PI3K bind different regions of the PDGFR. This possibility was also supported by the observation that the antibody to the PDGFR kinase insert Y751 region that blocks association of PI3K had only a minor effect on the in vitro binding of GAP. In addition, highly purified PI3K and GAP associated in the absence of other cellular proteins and neither cooperated nor competed with each other's binding to the PDGFR. Taken together, these studies indicate that GAP and PI3K bind directly to the PDGFR and have discrete binding sites that include portions of the kinase insert domain.     

Identification of major tyrosine phosphorylation sites in the human insulin receptor substrate Gab-1 by insulin receptor kinase in vitro

Gab-1 (Grb2-associated binder-1), which appears to play a central role in cellular growth response, transformation, and apoptosis, is a member of the insulin receptor substrate (IRS) family. IRS proteins act downstream in the signaling pathways of different receptor tyrosine kinases, including the insulin receptor (IR). In this paper, we characterize the phosphorylation of recombinant human Gab-1 (hGab-1) by IR in vitro. Kinetic phosphorylation data revealed that hGab-1 is a high affinity substrate for the IR (K(M): 12.0 microM for native IR vs 23.3 microM for recombinant IR). To elucidate the IR-specific phosphorylation pattern of hGab-1, we used phosphopeptide mapping by two-dimensional HPLC analysis. Phosphorylated tyrosine residues were subsequently identified by sequencing the separated phosphopeptides by matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) and Edman degradation. Our results demonstrate that hGab-1 was phosphorylated by IR at eight tyrosine residues (Y242, Y285, Y373, Y447, Y472, Y619, Y657, and Y689). Seventy-five percent of the identified radioactivity was incorporated into tyrosine residues Y447, Y472, and Y619 exhibiting features (NYVPM motif) of potential binding sites for the regulatory subunit (p85) of phosphatidylinositol (PI)-3 kinase. Accordingly, pull down assays with human HepG2 cell lysates showed that IR-specific phosphorylation of wild-type hGab-1 strongly enhanced PI-3 kinase binding. This is still the case when a single tyrosine residue in the NYVPM motif was mutated to phenylalanine. In contrast, phosphorylation-dependent binding of PI-3 kinase was completely abolished by changing a second tyrosine residue in a NYVPM motif independent from its location. Recently, we identified a similar cohort of tyrosine phosphorylation sites for the epidermal growth factor receptor (EGFR) with a predominant phosphorylation of tyrosine residue Y657 and binding of Syp [Lehr, S. et al. (1999) Biochemistry 38, 151-159]. These differences in the phosphorylation pattern of hGab-1 may contribute to signaling specificity by different tyrosine kinase receptors engaging distinct SH2 signaling molecules.     

Mutations of the platelet-derived growth factor receptor that cause a loss of ligand-induced conformational change, subtle changes in kinase activity, and impaired ability to stimulate DNA synthesis.

The wild-type and two mitogenic-defective mutants of the type beta receptor for platelet-derived growth factor (PDGF) were expressed in Chinese hamster ovary cells. In the first mutant, delta Ki, 82 of 104 amino acids in the kinase insert region were deleted. This mutant was recently reported to be defective in mediating DNA synthesis. In the second mutant, Y825F, tyrosine 825 was converted to phenylalanine by a point mutation. We report here that this mutant is also defective in mediating PDGF-stimulated DNA synthesis. Both mutants were capable of eliciting many of the early responses to PDGF, including receptor autophosphorylation. However, neither mutant was capable of undergoing PDGF-stimulated change in receptor conformation or of phosphorylating exogenous substrate in an in vitro assay. These data suggest that changes in receptor conformation and efficient utilization of specific tyrosine kinase substrates are important for the stimulation of cell proliferation of PDGF and that phosphorylation of tyrosine 825 may be involved in signal transduction.