Nontransforming Mutants Research Articles

JAK3 Mutants Transform Hematopoietic Cells through JAK1 Activation Causing T-Cell Acute Lymphoblastic Leukemia in a Bone Marrow Transplant Mouse Model

▪JAK3 is a cytosolic tyrosine kinase that associates with the common gamma chain in different cytokine receptors, in which the JAK1 kinase is another essential signaling protein. Large scale sequencing efforts recently identified mutations in the IL7R, JAK1 or JAK3 genes in about 25% of T-cell acute lymphoblastic leukemia (T-ALL) cases, with JAK3 being the most frequently (15% of T-ALL cases) mutated gene in this pathway. To determine if all mutations in JAK3 are true oncogenic mutations, we generated expression plasmids for 16 JAK3 mutants (M511I, A572T, A573V, R657Q, R657W, V674A, V678M, V678L, R775C, L857Q, Q865E, L875H, P906S, R925S, E958K, E1106G) and determined the in vitro and in vivo transforming properties, as well as their sensitivity to JAK kinase inhibitors.For 12 of 16 mutants expression in the IL3-dependent Ba/F3 cell line resulted in transformation to IL3 independent growth. Similarly, expression of the transforming mutants in IL7-receptor reconstituted 293T cells confirmed their ligand independent activation, while this was not observed for the non-transforming mutants. These 4 non-transforming mutants are likely to be passenger mutations, illustrating that results from sequencing always need to be confirmed by functional assays to distinguish driver mutations from passenger mutations. Most JAK3 mutants, except JAK3 L857Q and JAK3 L875H, were dependent on JAK1 kinase activity for their transforming capacities. In agreement with this, we observed that Ba/F3 cells transformed by the JAK1 dependent JAK3 mutants could be inhibited by ruxolitinib, a JAK1/JAK2 selective inhibitor, while the JAK3 L857Q and JAK3 L875H transformed cells were significantly less sensitive to ruxolitinib treatment. As expected, all JAK3 mutants were sensitive to the JAK3 selective inhibitor tofacitinib, except for JAK3 L875H, which showed resistance to all inhibitors tested.To determine the in vivo oncogenic properties of the JAK3 mutants, we expressed selected JAK3 mutants (M511I, A573V, L857Q, V674A and R657Q) in mouse hematopoietic cells through viral transduction. Mice transplanted with cells expressing JAK3 M511I, A573V or V674A showed a gradual increase of the WBC count and developed a T-ALL like disease within 120 to 200 days. In contrast, mice transplanted with cells expressing JAK3 L857Q or R657Q showed a lower increase in WBC count, and did present with severe splenomegaly and lymphadenopathy. Expression of JAK3 L857Q caused severe thymus hyperplasia, while the JAK3 R657Q mutant caused B-cell leukemia, illustrating that different JAK3 mutants seem to have variable oncogenic characteristics.Mice transplanted with cells expressing JAK3 M511I were treated with the JAK3 selective inhibitor tofacitinib and disease progression was followed by white blood cell count measurements. Treatment of the animals for 5 weeks with tofacitinib (oral gavage, 40 mg/kg/day) significantly decreased the disease progression compared to placebo treated mice. Moreover, we observed severe apoptosis of the leukemia cells in spleen and thymus in tofacitinib treated animals and not in placebo treated mice. However, tofacitnib treatment could not eradicate all leukemia cells, and the mice progressed when treatment was stopped.In conclusion, JAK3 is recurrently mutated in T-ALL patients, and we demonstrate that most JAK3 mutants are transforming proteins using In vitro and in vivo experiments. Our results show that JAK1 is an essential kinase for most JAK3 mutants, and that the majority of JAK3 mutants are sensitive to JAK3 and JAK1 specific inhibitors such as tofacitinib and ruxolitinib. However, some JAK3 mutants do show resistance to these inhibitors, which will need to be taken into account when trials are initiated for the treatment of ALL patients with JAK specific inhibitors. DisclosuresNo relevant conflicts of interest to declare.

Analysis of jaagsiekte sheep retrovirus (JSRV) envelope protein domains in transformation

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of a transmissible lung cancer in sheep. A unique feature is that JSRV envelope protein is also the oncogene for this virus. Previous studies have identified the cytoplasmic tail (CT) of the envelope transmembrane (TM) protein as critical for transformation although other regions of Env have also been implicated. In this study, the roles of other Env regions in transformation were investigated. Chimeras between JSRV Env and the Env of a related non-oncogenic endogenous retrovirus (enJSRV, 5F16) were used. A chimera containing the membrane-spanning region (MSR) of enJSRV inserted into JSRV Env showed substantially reduced transformation, indicating that the MSR plays a role in transformation. Transformation by this chimera was highly dependent on both Ras/Raf/MEK/MAPK and PI3K/Akt/mTOR signaling. A chimera containing the two amino acids in the TM ectodomain that distinguish JSRV and enJSRV showed modestly reduced transformation. Chimeras in the SU protein indicated that the amino terminal region of SU contributes to transformation, while the C-terminal part is not important. To test if Env trimerization is important for transformation, we mutated a leucine-rich sequence in the putative trimerization domain in the ectodomain of TM (Tri-M). This mutant could not transform cells and it did not oligomerize. However, Tri-M could complement a non-transforming mutant CT mutant (Y590F) so oligomerization is not necessary for at least some aspects of transformation. These experiments provide new insight into the regions and residues of JSRV Env protein necessary for oncogenic transformation.

Phosphorylation of histone H3 at Ser10: Its role in cell transformation by v-Src

We found that transformation by v-src constitutively activated phosphorylation of histone H3 at Ser10 in a transformation-specific manner. While nontransforming mutant of v-src did not activate H3 phosphorylation, H3 phosphorylation in cells expressing temperature-sensitive mutant of v-src was temperature-dependent. Inhibition of Ras signaling by Gap1m, a GTPase-activation protein for Ras, or S17N Ras, a dominant negative form of Ras, substantially suppressed the Ser10 phosphorylation of H3. Similarly, treatment of cells with manumycin A, a potent inhibitor of Ras-falnesyl transferase, clearly suppressed the H3 phosphorylation. In contrast, inhibition of STAT3 signaling or PI3K signaling did not perturb H3 phosphorylation. We found, however, inhibition of MEK or MSK1 markedly suppressed H3 phosphorylation. In addition, inhibition of MSK1 expression by its siRNA substantially suppressed H3 phosphorylation and anchorage-independent growth of transformed cells. Taken together, our results strongly suggest the importance of MSK1 and H3 phosphorylation in cell transformation by v-Src.

Polyoma and SV40 proteins differentially regulate PP2A to activate distinct cellular signaling pathways involved in growth control

Binding of Src family kinases to membrane-associated polyoma virus middle T-antigen (PyMT) can result in the phosphorylation of PyMT tyrosine 250, which serves as a docking site for the binding of Shc and subsequent activation of the Raf-MEK-ERK (MAP) kinase cascade. In a screen for PyMT variants that could not activate the ARF tumor suppressor, we isolated a cytoplasmic nontransforming mutant (MTA) that encoded a C-terminal truncated form of the PyMT protein. Surprisingly, MTA was able to strongly activate the MAP kinase pathway in the absence of Src family kinase and Shc binding. Interestingly, the polyoma small T-antigen (PyST), which shares with MTA both partial amino acid sequence homology and cellular location, also activates the MAP kinase cascade. Activation of the MAP kinase cascade by both MTA and PyST has been demonstrated to be PP2A-dependent. Neither MTA nor PyST activate the phosphorylation of AKT. The SV40 small T-antigen, which is similar to PyST in containing a J domain and in binding to the PP2A AC dimer, does not activate the MAP kinase cascade, but does stimulate phosphorylation of AKT in a PP2A-dependent manner. These findings highlight a novel role of PP2A in stimulating the MAP kinase cascade and indicate that the similar polyoma and SV40 small T-antigens influence PP2A to activate discrete cellular signaling pathways involved in growth control.

Interaction of the fork head domain transcription factor MPP2 with the human papilloma virus 16 E7 protein: enhancement of transformation and transactivation.

The high risk human papillomavirus (HPV) type 16 E7 protein affects cell growth control and promotes transformation by interfering with functions of cellular proteins. A key target of E7 is the tumor suppressor protein p105RB. Although this interaction is required for E7-dependent transformation, other cellular molecules must also be involved, because some E7 mutants that have reduced transforming abilities still bind to p105RB. In order to identify additional proteins that interact with E7 and that may be responsible to mediate its transforming function, we have used the C-terminal half of E7 in a yeast two-hybrid screen. We identified the fork head domain transcription factor M phase phosphoprotein 2 (MPP2) as an interaction partner of E7. Specific interaction of the two proteins both in vitro and in vivo in mammalian cells was detected. The interaction of MPP2 with E7 is functionally relevant since MPP2 enhances the E7/Ha-Ras co-transformation of rat embryo fibroblasts. In addition HPV16 E7, but neither non-transforming mutants of HPV16 E7 nor low risk HPV6 E7, was able to stimulate MPP2-specific transcriptional activity. Thus, MPP2 is a potentially important target for E7-mediated transformation.

Mutagenic analysis of Vav reveals that an intact SH3 domain is required for transformation.

The vav proto-oncogene encodes a protein with multiple modulae domains that enable it to function as a mediator, linking tyrosine signaling to downstream events in hematopoietic cells. Circumstantial evidence suggests that protein-protein interactions exerted by two of these domains, the Src homology 2 (SH2) and the Src homology 3 (SH3), play an important role in the regulation of Vav activity. To study the relevance of the SH3 domain for the function of vav as a transforming gene, we have created several mutations in the SH3 domain located at its carboxy region. Substitution of the non-conserved aspartic acid 797 (to asparagine, D797N) retained the transforming potential of the vav oncogene, whereas substitutions of five highly conserved amino-acids: alanine 789 (to asparagine, A789N), leucine 801 (to arginine, L801R), tryptophan 821 (to arginine, W821R), glycine 830 (to valine, G830V) and valine 837 (to glutamic acid, V837E) greatly reduced its transforming potential. The mutant proteins resemble Vav in many biochemical properties; however, while the transforming mutant protein (D797N) associates with several unidentified proteins in a manner similar to that of Vav, the non-transforming mutant Vav proteins react very poorly with these proteins. Among the known Vav-interacting proteins, hnRNP-K associates with all mutant proteins except A789N and V837E whereas binding of Zyxin to any of the mutant proteins is not affected. Taken together, our results clearly demonstrate that the SH3 domain has a positive effect on vav activity and is needed for vav transformation. The vavSH3C associating protein(s) that are crucial for its activity as a transforming gene have probably not yet been identified.

A transformation-associated complex involving tyrosine kinase signal adapter proteins and caldesmon links v-erbB signaling to actin stress fiber disassembly.

The avian erythroblastosis viral oncogene (v-erbB) encodes a receptor tyrosine kinase that possesses sarcomagenic and leukemogenic potential. We have expressed transforming and nontransforming mutants of v-erbB in fibroblasts to detect transformation-associated signal transduction events. Coimmunoprecipitation and affinity chromatography have been used to identify a transformation-associated, tyrosine phosphorylated, multiprotein complex. This complex consists of Src homologous collagen protein (Shc), growth factor receptor binding protein 2 (Grb2), son of sevenless (Sos), and a novel tyrosine phosphorylated form of the cytoskeletal regulatory protein caldesmon. Immunofluorescence localization studies further reveal that, in contrast to the distribution of caldesmon along actin stress fibers in normal fibroblasts, caldesmon colocalizes with Shc in plasma membrane blebs in transformed fibroblasts. This colocalization of caldesmon and Shc correlates with actin stress fiber disassembly and v-erbB-mediated transformation. The tyrosine phosphorylation of caldesmon, and its association with the Shc-Grb2-Sos signaling complex directly links tyrosine kinase oncogenic signaling events with cytoskeletal regulatory processes, and may define one mechanism regulating actin stress fiber disassembly in transformed cells.

Characterization of changes in gene expression associated with malignant transformation by the NF-kappaB family member, v-Rel.

In this study, alterations in gene expression patterns have been examined in v-Rel-transformed avian bone marrow cells. Using a conditional v-Rel estrogen receptor chimera (v-RelER) which transforms cells in an estrogen-dependent manner, we constructed subtraction cDNA libraries from v-RelER-transformed bone marrow cells. Several different sequences were identified whose expression was altered upon hormone activation of v-RelER. These include two genes related to the MIP-1 chemokine family (mip-1beta and a tca3 homologue), a cell surface antigen sca-2 and the transcription factor nfkb1. The expression of each gene was assayed in a number of wild-type and mutant v-Rel-expressing fibroblast and hematopoietic cells. All v-Rel-transformed hematopoietic cells tested express high levels of nfkb1 and sca-2. In fibroblasts, wild-type v-Rel induced expression of mip-1beta and nfkb1, while nontransforming mutants of v-Rel failed to do so, suggesting a role for these two genes in v-Rel mediated transformation. Finally, these genes are expressed at high levels in cells overexpressing wild-type and truncated forms of c-Rel, implying that v-Rel transforms, in part, by induction of c-Rel target genes.

Comparison of the low energy conformations of an oncogenic and a non-oncogenic p21 protein, neither of which binds GTP or GDP

Oncogenic p21 protein, encoded by the ras-oncogene, that causes malignant transformation of normal cells and many human tumors, is almost identical in sequence to its normal protooncogene-encoded counterpart protein, except for the substitution of arbitrary amino acids for the normally occurring amino acids at critical positions such as Gly 12 and Gln 61. Since p21 is normally activated by the binding of GTP in place of GDP, it has been postulated that oncogenic forms must retain bound GTP for prolonged time periods. However, two multiply substituted p21 proteins have been cloned, neither of which binds GDP or GTP. One of these mutant proteins with Val for Gly 10, Arg for Gly 12, and Thr for Ala 59 causes cell transformation, while the other, similar protein with Gly 10, Arg 12, Val for Gly 13 and Thr 59 does not transform cells. To define the critical conformational changes that occur in the p21 protein that cause it to become oncogenic, we have calculated the low energy conformations of the two multiply substituted mutant p21 proteins using a new adaptation of the electrostatically driven Monte Carlo (EDMC) technique, based on the program ECEPP. We have used this method to explore the conformational space available to both proteins and to compute the average structures for both using statistical mechanical averaging. Comparison of the average structures allows us to detect the major differences in conformation between the two proteins. Starting structures for each protein were calculated using the recently deposited x-ray crystal coordinates for the p21 protein, that was energy-refined using ECEPP, and then perturbed using the EDMC method to compute its average structure. The specific amino acid substitutions for both proteins were then generated into the lowest energy structure generated by this procedure, subjected to energy minimization and then to full EDMC perturbations. We find that both mutant proteins exhibit major differences in conformation in specific regions, viz., residues 35-47, 55-78, 81-93, 96-110, 115-126, and 123-134, compared with the EDMC-refined x-ray structure of the wild-type protein. These regions have been found to be the most flexible in the p21 protein bound to GDP from prior molecular dynamics calculations (Dykes et al., 1993). Comparison of the EDMC-average structure of the transforming mutant with that of the nontransforming mutant reveals major structural differences at residues 10-16, 32-40, and 60-68. These structural differences appear to be the ones that are critical in activation of the p21 protein.(ABSTRACT TRUNCATED AT 400 WORDS)

All three domains of the Epstein-Barr virus-encoded latent membrane protein LMP-1 are required for transformation of rat-1 fibroblasts

LMP-1, the Epstein-Barr virus latent membrane protein 1, is the only protein encoded by the virus that has been shown to have the properties of a transforming oncogene in rodent fibroblasts such as Rat-1 cells. LMP-1 is phosphorylated and proteolytically cleaved in Rat-1 cells in a manner similar to that seen in human lymphocytes. In this study, we demonstrate that all three major domains of LMP-1 (N-terminal, transmembrane, and C-terminal domains) are required for the ability to transform Rat-1 cells in culture, as assayed by loss of contact inhibition. This study is the first demonstration of a functional role for the C-terminal domain of LMP-1. Our analysis suggests that there are at least three distinct regions of the C terminus involved in signalling. Amino acids 306 to 334, which generate a toxic signal in the absence of amino acids 334 to 364, and the last 23 amino acids, 364 to 386, are essential for transformation. Biochemical analysis of the LMP-1 mutants with the three domains deleted indicate that the mutant N-terminal with the domain deleted is phosphorylated normally but is inefficiently cleaved compared with the wild-type LMP-1. The mutant with the transmembrane domain deleted is also phosphorylated but is not cleaved, showing that phosphorylation of LMP-1 does not require membrane association. The nontransforming mutant with the C-terminal domain deleted that lacks the last 23 amino acids is phosphorylated and cleaved. Therefore, these processing events alone are insufficient to generate a transforming signal.

Phosphatidylinositol Kinase Activity Associates with Viral p60src Protein

Immunoprecipitates of p60v-src proteins from chicken embryo fibroblasts infected with Rous sarcoma virus were assayed for phosphatidylinositol (PI) kinase activity in the absence of detergents. The product of the PI kinase reaction, phosphatidylinositol monophosphate (PIP), migrated slightly slower than did the authentic phosphatidylinositol-4-monophosphate marker in thin-layer chromatography and was indistinguishable from phosphatidylinositol-3-monophosphate produced by PI kinase type I. Furthermore, the deacylated product comigrated with glycerophosphoinositol-3-phosphate in high-performance liquid chromatography. Both sucrose gradient fractionation and the heat stability of PI kinase activity from cells infected with temperature-sensitive mutants suggest that the PI kinase activity is not intrinsic to p60v-src but is a property of another molecule complexed with p60v-src. All transforming variants of p60src were associated with PI kinase activity, whereas this enzyme activity was hardly detectable in immunoprecipitates from cells infected with nontransforming viruses encoding p60c-src or an enzymatically inactive variant. However, PI kinase activity was found in p60src immunoprecipitates from cells infected with nonmyristylated, nontransforming mutants as well as temperature-sensitive mutants at the nonpermissive temperature, which indicated that simple association of PI kinase activity with p60src is not sufficient for cell transformation.

Phosphatidylinositol kinase activity associates with viral p60src protein.

Immunoprecipitates of p60v-src proteins from chicken embryo fibroblasts infected with Rous sarcoma virus were assayed for phosphatidylinositol (PI) kinase activity in the absence of detergents. The product of the PI kinase reaction, phosphatidylinositol monophosphate (PIP), migrated slightly slower than did the authentic phosphatidylinositol-4-monophosphate marker in thin-layer chromatography and was indistinguishable from phosphatidylinositol-3-monophosphate produced by PI kinase type I. Furthermore, the deacylated product comigrated with glycerophosphoinositol-3-phosphate in high-performance liquid chromatography. Both sucrose gradient fractionation and the heat stability of PI kinase activity from cells infected with temperature-sensitive mutants suggest that the PI kinase activity is not intrinsic to p60v-src but is a property of another molecule complexed with p60v-src. All transforming variants of p60src were associated with PI kinase activity, whereas this enzyme activity was hardly detectable in immunoprecipitates from cells infected with nontransforming viruses encoding p60c-src or an enzymatically inactive variant. However, PI kinase activity was found in p60src immunoprecipitates from cells infected with nonmyristylated, nontransforming mutants as well as temperature-sensitive mutants at the nonpermissive temperature, which indicated that simple association of PI kinase activity with p60src is not sufficient for cell transformation.

Relationship among guanine nucleotide exchange, GTP hydrolysis, and transforming potential of mutated ras proteins.

The effect of a series of mutations on the transforming potential of normal human rasH has been compared with their effects on GTPase and guanine nucleotide exchange rates of p21. The mutation Val-146 resulted in partial activation of transforming potential which could be attributed to a greater than 1,000-fold-increased rate of nucleotide exchange in the absence of an effect on GTPase. In contrast, the more modest enhancement of exchange rate (approximately 100-fold) which resulted from the mutation Met-14 did not affect biological activity. The partially activating mutation Thr-59 was found to result in both a 5-fold reduction in GTPase and a 10-fold increase in nucleotide exchange. However, the nontransforming mutant Ile-59 displayed a comparable decrease in GTPase without an effect on nucleotide exchange. The activating effect of the Thr-59 mutation may thus represent a combined effect of reduced GTPase and increased exchange. Similarly, the strongly activating mutation Leu-61 resulted in a fivefold increase in nucleotide exchange in addition to decreased GTPase, whereas weakly activating mutations at position 61 (Trp and Pro) resulted only in decreased GTPase without affecting nucleotide exchange rates. Finally, combining the two mutations Met-14 and Ile-59, which alone had no effect on biological activity, yielded a double mutant with a 20-fold increased transforming potential, demonstrating a synergistic effect of these two mutations. Overall, these results indicate that large increases in nucleotide exchange can activate ras transforming potential in the absence of decreased GTPase and that relatively modest increases in nucleotide exchange can act synergistically with decreased GTPase to contribute to ras activation.

Relationship among guanine nucleotide exchange, GTP hydrolysis, and transforming potential of mutated ras proteins.

The effect of a series of mutations on the transforming potential of normal human rasH has been compared with their effects on GTPase and guanine nucleotide exchange rates of p21. The mutation Val-146 resulted in partial activation of transforming potential which could be attributed to a greater than 1,000-fold-increased rate of nucleotide exchange in the absence of an effect on GTPase. In contrast, the more modest enhancement of exchange rate (approximately 100-fold) which resulted from the mutation Met-14 did not affect biological activity. The partially activating mutation Thr-59 was found to result in both a 5-fold reduction in GTPase and a 10-fold increase in nucleotide exchange. However, the nontransforming mutant Ile-59 displayed a comparable decrease in GTPase without an effect on nucleotide exchange. The activating effect of the Thr-59 mutation may thus represent a combined effect of reduced GTPase and increased exchange. Similarly, the strongly activating mutation Leu-61 resulted in a fivefold increase in nucleotide exchange in addition to decreased GTPase, whereas weakly activating mutations at position 61 (Trp and Pro) resulted only in decreased GTPase without affecting nucleotide exchange rates. Finally, combining the two mutations Met-14 and Ile-59, which alone had no effect on biological activity, yielded a double mutant with a 20-fold increased transforming potential, demonstrating a synergistic effect of these two mutations. Overall, these results indicate that large increases in nucleotide exchange can activate ras transforming potential in the absence of decreased GTPase and that relatively modest increases in nucleotide exchange can act synergistically with decreased GTPase to contribute to ras activation.

Different patterns of Epstein-Barr virus gene expression and of cytotoxic T-cell recognition in B-cell lines infected with transforming (B95.8) or nontransforming (P3HR1) virus strains.

Epstein-Barr virus (EBV)-negative Burkitt's lymphoma (BL) cell lines have been converted to EBV genome positivity by in vitro infection with the transforming EBV strain B95.8 and with the nontransforming mutant strain P3HR1, which has a deletion in the gene encoding the nuclear antigen EBNA2. These B95.8- and P3HR1-converted lines have been compared for their patterns of expression of EBV latent genes (i.e., those viral genes constitutively expressed in all EBV-transformed lines of normal B-cell origin) and for their recognition by EBV-specific cytotoxic T lymphocytes (CTLs), in an effort to identify which latent gene products provide target antigens for the T-cell response. B95.8-converted lines on several different EBV-negative BL-cell backgrounds all showed detectable expression of the nuclear antigens EBNA1, EBNA2, and EBNA3 and of the latent membrane protein (LMP); such converts were also clearly recognized by EBV-specific CTL preparations with restriction through selected human leukocyte antigen (HLA) class I antigens on the target cell surface. The corresponding P3HR1-converted lines (lacking an EBNA2 gene) expressed EBNA1 and EBNA3 but, surprisingly, showed no detectable LMP; furthermore, these converts were not recognized by EBV-specific CTLs. Such differences in T-cell recognition were not due to any differences in expression of the relevant HLA-restricting determinants between the two types of convert, as shown by binding of specific monoclonal antibodies and by the susceptibility of both B95.8 and P3HR1 converts to allospecific CTLs directed against these same HLA molecules. The results suggest that in the normal infectious cycle, EBNA2 may be required for subsequent expression of LMP and that both EBNA2 and LMP (but not EBNA1 or EBNA3) may provide target antigens for the EBV-specific T-cell response.

Interactions between polyomavirus medium T antigen and three cellular proteins of 88, 61, and 37 kilodaltons.

Affinity-purified medium T antigen of wild-type polyomavirus and dl8, a transforming mutant with a deletion in the medium T gene, is associated with three cellular proteins with apparent molecular weights of 88,000 (88K protein), 61,000 (61K protein), and 37,000 (37K protein). Medium T antigen encoded by the nontransforming hrt mutants fails to associate with these proteins, whereas medium T antigen of the nontransforming mutant dl1015 is able to do so. Medium T antigen of the nontransforming mutant dl23 binds to the 61K and 37K proteins; however, binding to the 88K protein is uncertain. The pattern of complex formation between these proteins and medium T antigen resembles that of pp60c-src and medium T antigen. The binding of medium T antigen to the 88K, 61K, and 37K proteins, as well as to pp60c-src, might represent a necessary but insufficient step in transformation. By mixing extracts from infected and uninfected cells, complex formation between medium T antigen and the 88K, 61K, and 37K proteins can be demonstrated in vitro. Pulse-chase experiments indicated that in vivo the association between medium T antigen and the 61K and 37K proteins is a slow process. The latter two proteins are probably bound to each other in uninfected cells. On two-dimensional gels of whole-cell extract, the 61K protein comigrated with a minor protein with an isoelectric point of 5.2. The 61K protein was neither phosphorylated nor glycosylated. Polyomavirus tumor serum precipitated the 61K and 37K proteins independently of medium T antigen. Therefore, the 61K protein or the 37K protein or both have the properties of a cellular tumor antigen.

An 81 kd protein complexed with middle T antigen and pp60 c- src: A possible phosphatidylinositol kinase

It has previously been shown that a proportion of middle T antigen molecules exist in a stable complex with pp60 c- src . Here we show that there appears to be a third component to the complex, a protein of molecular mass 81 kd (p81). p81 was phosphorylated exclusively on tyrosine residues in kinase assays performed using immunoprecipitates from polyoma virus-transformed cells and antibodies to both middle T and pp60 c- src , and was also detected when immunoprecipitates were made from lysates of 32P-labeled cells. p81 was bound to middle T and pp60 c- src in cell lines containing transforming mutants of middle T, but not (in phosphorylated form) to all nontransforming mutants. A parallel investigation of phosphatidylinositol kinase activity in immune complexes containing these middle T mutants revealed a complete coincidence between the presence of p81 and phosphatidylinositol kinase activity. We therefore suggest that p81 is a phosphatidylinositol kinase.

Medium tumor antigen of polyomavirus transformation-defective mutant NG59 is associated with 73-kilodalton heat shock protein.

Affinity-purified medium T antigen encoded by NG59, a nontransforming mutant of polyomavirus, is specifically associated with a protein of 72,000 daltons (72K protein). Medium T antigens of wild-type polyomavirus and the transformation-competent mutant dl8 are not associated with the 72K protein. Instead, they form a complex with another protein of 61,000 daltons. Several lines of evidence suggest that the medium T antigen-associated 72K protein is equivalent to the abundant and constitutive 73K heat shock protein. First, on two-dimensional polyacrylamide gels the 72K protein migrated with the same pI (5.6) as did the 73K heat shock protein. Second, the 72K protein was immunoprecipitable with antibodies against heat shock proteins. Third, when digested with V8 protease, the 72K protein gave rise to the same pattern of fragments as did the 73K heat shock protein.

In vitro mutagenesis of the v-sis transforming gene defines functional domains of its growth factor-related product.

The polypeptide sequence of the v-sis transforming gene product of simian sarcoma virus (SSV) can be divided into four regions that are likely to represent structural domains of the protein. Mutations were generated in the SSV nucleotide sequence to assay the extent or function of each of these regions. The results indicate that the helper virus-derived amino-terminal sequence as well as a core region homologous to polypeptide chain 2 of platelet-derived growth factor (PDGF) are required for the transforming function of the protein. Products of transforming but not nontransforming mutants formed dimer structures conformationally analogous to biologically active PDGF.

Characterization of viable mutants of polyomavirus cold sensitive for maintenance of cell transformation

We mutagenized a cloned fragment of polyoma DNA encoding portions of the middle size (MT) and large T antigens. We regenerated infectious viral genomes containing the mutagenized DNA and tested their transforming ability at 32 and 39 degrees C. We isolated three nontransforming mutants and two mutants which were cold sensitive for the maintenance of cell transformation. The nontransforming mutants contained amber termination codons in the reading frame for the MT antigen. They synthesized truncated MT antigens which lacked MT-associated protein kinase activity. The cold-sensitive mutants synthesized MT antigens indistinguishable from wild type with regard to size, stability at 32 and 39 degrees C, intracellular location, and associated protein kinase activity. One of the mutants was shown by nucleotide sequence analysis to contain a single amino acid change in the MT antigen, located two residues upstream from the C-terminal hydrophobic region, and no changes in the large T antigen. The other mutant contained two amino acid changes in the MT antigen and two amino acid changes in the large T antigen.