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.
D000230:Adenocarcinoma / Q000201:enzymology*; D002021:Buffers; D002478:Cells, Cultured; D003110:Colonic Neoplasms / Q000201:enzymology*; D006801:Humans; D007158:Immunologic Techniques; D007413:Intestinal Mucosa / Q000201:enzymology*; D015688:Oncogene Protein pp60(v-src); D010449:Peptide Mapping; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D019000:Phosphotyrosine; D017027:Protein Tyrosine Phosphatases; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D012191:Retroviridae Proteins / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology
D002021:Buffers; D012191:Retroviridae Proteins; D019000:Phosphotyrosine; D014638:Vanadates; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D015688:Oncogene Protein pp60(v-src); D010749:Phosphoprotein Phosphatases; D017027:Protein Tyrosine Phosphatases