Pp60c-src Protein Kinase Activity Research Articles

Pp60c‐src protein kinase activity in human gastric carcinomas

We examined pp60c-src protein kinase activity in human gastric carcinoma cell lines and gastric carcinoma tissues as well as normal mucosa. pp60c-src kinase activity was detected in all 5 carcinoma cell lines at various levels. Of 16 gastric carcinoma tissues, 8 showed higher pp60c-src kinase activity in tumor tissues than in corresponding normal mucosa. However, the levels of expression of pp60c-src detected by Western blotting were not always consistent with the activities of pp60c-src protein kinase. These findings suggest that the increase in pp60c-src protein kinase activity might be brought about by post-translational changes.

Activation of the pp60c-src protein kinase is an early event in colonic carcinogenesis.

Colonic neoplasia provides an opportunity to study tumor progression because most carcinomas arise from adenomas (polyps), which, in turn, arise from normal epithelia. The malignant potential of adenomas varies with size, histology, and degree of dysplasia. Polyps that are less than 2 cm with villous architecture and severe dysplasia are most likely to contain carcinoma. Previous studies demonstrated that the in vitro protein-tyrosine kinase activity of pp60c-src from colon carcinomas is significantly higher than that from adjacent normal mucosa. Here we report that the protein kinase activity of pp60c-src is also elevated in colonic polyps. Activity is highest in malignant polyps and in greater than 2-cm benign polyps that contain villous structure and severe dysplasia. Thus, pp60c-src activation occurs in benign polyps that are at greatest risk for developing cancer. These data suggest that activation of the protooncogene product pp60c-src may be an important event in the genesis of human colon carcinoma.

Biological and Biochemical Properties of the c-src+ Gene Product Overexpressed in Chicken Embryo Fibroblasts

The c-src protein isolated from neuronal cells (pp60c-src+) displays a higher level of protein kinase activity than does pp60c-src from nonneural tissues. There are two structural alterations present in the amino-terminal half of pp60c-src+ expressed in neurons which could contribute to the enhanced activity of this form of pp60c-src: (i) a hexapeptide insert located at amino acid 114 of avian pp60c-src+ and (ii) a novel site(s) of serine phosphorylation. We characterized pp60c-src+ expressed in a nonneuronal cell type to identify factors that regulate the activity of the c-src+ protein and the importance of the neuronal environment on this regulation. The c-src+ protein overexpressed in chicken embryo fibroblasts (CEFs) displayed higher kinase activity than did pp60c-src. The major sites of phosphorylation of the c-src+ protein were Ser-17 and Tyr-527. The unique site(s) of serine phosphorylation originally identified in pp60c-src+ expressed in neurons was not detected in the c-src+ protein overexpressed in CEFs. Therefore, the hexapeptide insert is sufficient to cause an elevation in the tyrosine protein kinase activity of pp60c-src+. Our data also indicate that CEFs infected with the Rous sarcoma virus (RSV)c-src+ display phenotypic changes that distinguish them from cultures producing pp60c-src and that pp60c-src+-expressing cells are better able to grow in an anchorage-independent manner. The level of total cellular tyrosine phosphorylation in RSVc-src+-infected cultures was moderately higher than the level observed in cultures infected with RSVc-src. This level was not as pronounced as that observed in cells infected with RSVv-src or oncogenic variants of RSVc-src. Thus, pp60c-src+ could be considered a partially activated c-src variant protein much like other c-src proteins that contain mutations in the amino-terminal domain.

Biological and biochemical properties of the c-src+ gene product overexpressed in chicken embryo fibroblasts.

The c-src protein isolated from neuronal cells (pp60c-src+) displays a higher level of protein kinase activity than does pp60c-src from nonneural tissues. There are two structural alterations present in the amino-terminal half of pp60c-src+ expressed in neurons which could contribute to the enhanced activity of this form of pp60c-src: (i) a hexapeptide insert located at amino acid 114 of avian pp60c-src+ and (ii) a novel site(s) of serine phosphorylation. We characterized pp60c-src+ expressed in a nonneuronal cell type to identify factors that regulate the activity of the c-src+ protein and the importance of the neuronal environment on this regulation. The c-src+ protein overexpressed in chicken embryo fibroblasts (CEFs) displayed higher kinase activity than did pp60c-src. The major sites of phosphorylation of the c-src+ protein were Ser-17 and Tyr-527. The unique site(s) of serine phosphorylation originally identified in pp60c-src+ expressed in neurons was not detected in the c-src+ protein overexpressed in CEFs. Therefore, the hexapeptide insert is sufficient to cause an elevation in the tyrosine protein kinase activity of pp60c-src+. Our data also indicate that CEFs infected with the Rous sarcoma virus (RSV)c-src+ display phenotypic changes that distinguish them from cultures producing pp60c-src and that pp60c-src+-expressing cells are better able to grow in an anchorage-independent manner. The level of total cellular tyrosine phosphorylation in RSVc-src+-infected cultures was moderately higher than the level observed in cultures infected with RSVc-src. This level was not as pronounced as that observed in cells infected with RSVv-src or oncogenic variants of RSVc-src. Thus, pp60c-src+ could be considered a partially activated c-src variant protein much like other c-src proteins that contain mutations in the amino-terminal domain.

Pp60c-src expression in the developing rat brain.

We have studied pp60c-src expression in the striatum, hippocampus, and cerebellum of the developing rat brain. In the striatum, pp60c-src protein kinase activity peaks during embryonic development and then declines in the adult. The peak activity occurs in the striatum on embryonic day 20 (E20) when it is 18- to 20-fold higher than the activity in fibroblasts and 4- to 5-fold higher than the activity in the striatum at E15 or in the adult striatum. In the hippocampal region, pp60c-src activity reaches a maximum shortly after birth but remains high throughout life. On postnatal day 2 (P2) the activity in the hippocampus is 9- to 13-fold higher than the activity in fibroblasts and twice as high as the activity in the hippocampus at E18. In the cerebellum, the kinase activity remains constant from E20 onward and is 6- to 10-fold higher than that observed in fibroblasts. The increase in pp60c-src kinase activity observed during the development of the striatum and hippocampus is due to an increase in the amount of pp60c-src protein and to an increase in the specific activity of the kinase. The increase in specific activity in these regions coincides with the peak periods of neurogenesis and neuronal growth. In the striatum, we have found that the increase in pp60c-src activity also parallels the increase observed in culture as embryonic striatal neurons differentiate. Taken together, our results are consonant with the idea that pp60c-src is the product of a developmentally regulated gene that is important for the differentiation and/or the continuing function of neurons.

Analysis of pp60c‐src tyrosine kinase activity and phosphotyrosyl phosphatase activity in human colon carcinoma and normal human colon mucosal cells

We have compared the level of phosphotyrosyl phosphatase activity in lysates from normal human colon mucosal cells and human colon carcinoma cells and analyzed the effect of incubating these cells with sodium orthovanadate, an inhibitor of phosphotyrosyl phosphatase activity, on the relative abundance of acid-stable phosphotyrosine and on in vitro protein kinase activity of pp60c-src. Additionally, we compared the effect of lysing these cells in buffer containing only nonionic detergents with RIPA buffer, which contains both sodium dodecyl sulfate and deoxycholate, on the in vitro kinase activity of pp60c-src. Our results show that the level of detectable phosphotyrosyl phosphatase activity in lysates derived from normal colon cells and colon carcinoma cells is very similar. Additionally, the abundance of acid-stable phosphotyrosine in these cells cultured in the absence or presence of vanadate is not significantly different. However, incubation of these cells with vanadate significantly stimulates the activity of pp60c-src derived from the normal colon cells in immune-complex kinase assays, while having no detectable effect on the activity of pp60c-src from the colon tumor cells. The in vitro protein kinase activity of pp60c-src derived from RIPA buffer lysates of colon carcinoma cells was found to be elevated five- to sevenfold when compared with pp60c-src from these same cells lysed in buffer containing only Nonidet-P 40 as a detergent. The type of lysis buffer did not effect the activity of pp60c-src from normal colon mucosal cells. These results provide additional evidence that the activity of pp60c-src may be regulated differently in colon carcinoma and normal colon mucosal cells.

Activation of pp60c-src protein kinase activity in human colon carcinoma.

The tyrosine-specific protein kinase activity of pp60c-src molecules obtained from human colon carcinoma tissues and tumor-derived cell lines was found to be elevated over that from normal colon tissues or cultures of normal colon mucosal cells. The elevated pp60c-src protein kinase activity in tumor tissues and in cultured colon carcinoma cells does not appear to result solely from an increase in the abundance of the c-src-encoded protein, suggesting that the specific activity of the pp60c-src tyrosine phosphotransferase is enhanced. These results raise the possibility that activation of the pp60c-src protein kinase may contribute to the genesis of human colon tumors.

Analysis of polyomavirus middle-T-antigen-transformed rat cell variants expressing different levels of pp60c-src

We characterize two independent variant cellular clones which arose following in vitro passage of polyomavirus middle-T-antigen (MTAg)-transformed FR3T3 cells expressing RNA complementary to c-src mRNA. These clones were initially flat and underwent morphologic transformation at a high frequency to a phenotype indistinguishable from that of parental MTAg-transformed FR3T3 cells. Biochemical analysis of the flat clones prior to phenotypic conversion revealed that these cells synthesized little detectable pp60c-src and had correspondingly low levels of pp60c-src protein kinase activity and MTAg-associated protein kinase activity. The flat cell clones did not possess detectable focus-forming activity, were not capable of detectable anchorage-independent growth, and had saturation densities and doubling times below those normally observed for FR3T3 cells. Following conversion of the flat clones to a shape resembling that of typical MTAg-transformed cells, the abundance of pp60c-src, pp60c-src kinase activity, and MTAg-associated in vitro protein kinase activity were all restored to the levels found in the parental MTAg transformants. These cells had growth rates, focus-forming activities, anchorage-independent growth rates, and saturation densities similar to those of the parental MTAg-transformed rat cells. These data provide additional evidence that maintenance of a transformed phenotype by polyomavirus MTAg in established rat cell lines depends, at least in part, on a minimal threshold level of pp60c-src.

Analysis of pp60c-src protein kinase activity in human tumor cell lines and tissues.

We have evaluated the level of pp60c-src protein kinase activity in a variety of human tumor tissues and human tumor cell lines, and have estimated the abundance of the c-src protein in several of these tissues and cell lines. All cell lines derived from tumors of neuroectodermal origin that express a neural phenotype were found to possess c-src molecules with high levels of tyrosine-specific protein kinase activity. In contrast, cell lines derived from tumors of neuroectodermal origin that do not express neural characteristics, such as glioblastomas and melanomas, were found to have pp60c-src molecules with low levels of protein kinase activity. A similar pattern was observed when we analyzed the activity of c-src molecules extracted directly from corresponding tumor tissues. Analysis of human tumor cell lines derived from tissues other than those of neuroectodermal origin revealed that pp60c-src protein kinase activity was low in most cases. Exceptions to this observation were all rhabdomyosarcoma, osteogenic sarcoma, Ewing's sarcoma, and colon carcinoma lines tested. Comparison of pp60c-src kinase activity in normal skeletal muscle and rhabdomyosarcoma tissue and in normal breast tissue and breast adenocarcinoma tissue revealed that pp60c-src kinase activity was specifically elevated in the tumor tissues in both cases. However, the amount of pp60c-src protein in both normal and tumor tissues was found to be similar. These observations suggest that increases in the specific activity of the pp60c-src phosphotransferase in some rhabdomyosarcomas and breast carcinomas may be a characteristic acquired during the malignant transformation of the cells that is retained in cell lines established from these tumors.

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.

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.

Analysis of pp60c-src protein kinase activity in hamster embryo cells transformed by simian virus 40, human adenoviruses, and bovine papillomavirus 1

We have examined the effect of DNA tumor virus transformation of primary hamster embryo cells on the tyrosyl kinase activity of pp60c-src. Our present study demonstrates that some clones of hamster embryo cells transformed by simian virus 40, adenovirus type 2, adenovirus type 12, or bovine papillomavirus 1 can possess elevated pp60c-src kinase activity when compared with normal hamster embryo cells. However, other clones of hamster embryo cells transformed by these same viruses were found to have normal levels of pp60c-src kinase activity. In those clones of transformed cells where pp60c-src kinase activity was elevated, the increased levels of kinase activity were the result of an apparent increase in the specific activity of the pp60c-src phosphotransferase rather than an increase in the amount of the src gene product. Additionally, pp60c-src was not found to be physically associated with tumor antigens known to be encoded by these viruses. These results indicate that elevated levels of pp60c-src kinase activity can be found in hamster embryo cells transformed by several different DNA tumor viruses and suggest that the molecular mechanism by which pp60c-src kinase activity is elevated may differ from that previously observed in polyomavirus-transformed cells. These results also imply that elevation of pp60c-src kinase activity is not required for the transformation of hamster cells by these viruses.

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.

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.