Characterization of the protein apparently responsible for the elevated tyrosine protein kinase activity in LSTRA cells.

The LSTRA murine thymoma cell line contains an elevated level of tyrosine protein kinase activity. When a microsomal preparation from these cells is incubated in vitro with ATP, the principal tyrosine protein kinase substrate is a 56,000-dalton protein, p56. We have found that an activity phosphorylating p56 on tyrosine can also be detected at low levels in microsomes from most, but not all, T lymphoma cell lines and from normal thymic tissue. Only 1 of 30 other lymphoma cell lines was found to contain an elevated level of such a tyrosine protein kinase. An activity that phosphorylated p56 in vitro was not detectable in the cells of other hematopoietic lineages. Anti-peptide antibodies reactive with the site of in vitro tyrosine phosphorylation of p56 allowed us to determine that the apparent abundance of the p56 polypeptide parallels closely the level of the tyrosine protein kinase activity in the cell lines examined. This suggests that p56 is the protein kinase responsible for the elevated tyrosine protein kinase activity in LSTRA cells and that the phosphorylation of p56 observed in vitro results from autophosphorylation. Two-dimensional tryptic peptide mapping revealed that p56 is distinct from the proteins encoded by the cellular genes which are the progenitors of retroviral tyrosine protein kinases, src, yes, fgr, abl, fes, and ros. Additionally, none of these proto-oncogenes was found to be transcribed at elevated levels in LSTRA or Thy19 cells. Like the catalytic subunit of the cyclic AMP-dependent protein kinase, the cellular and viral forms of p60src, and the protein phosphatase calcineurin B, p56 contains covalently bound fatty acid.     

Identification of the human T-lymphocyte protein-tyrosine kinase by peptide-specific antibodies

We have recently cloned a cDNA from the human T-cell leukemia, JURKAT, having homology with the src-like family of protein-tyrosine kinases. We have made rabbit polyclonal antibodies against the synthetic peptide CKERPEDRPTFDYLRSVLEDFFTATEGQYQPQP (cys-33-pro) deduced from the carboxy-terminal amino acid sequence predicted by the JURKAT cDNA. In this report, we demonstrate that these antibodies immunoprecipitate the protein-tyrosine kinase activity from solubilized membrane extracts from JURKAT T-leukemia cells and from human peripheral blood T-lymphocytes from normal donors. A 58 kd protein, exhibiting protein-tyrosine kinase activity, was specifically immunoprecipitated in both cases. The antibodies failed to crossreact with pp60c-src from human platelets, but did crossreact with the murine T-lymphocyte protein-tyrosine kinase, pp56T-cell.     

High level of tyrosine protein kinase in a murine lymphoma cell line induced by Moloney leukemia virus.

Several sarcoma-inducing viruses encode protein kinases that phosphorylate tyrosine residues. Such enzymatic activities can be detected within the detergent-insoluble matrix of transformed fibroblasts. We have analysed the protein kinase activities in two murine lymphoma cell lines ( MBL2 and LSTRA) induced by Moloney murine leukemia virus (Mo-MuLV). After incubation of the detergent-insoluble matrix of these cells with [gamma-32P]ATP, several alkali-resistant phosphoproteins, including a very heavily labelled 55 000 mol. wt. protein ( p55 ), have been detected in LSTRA, reflecting the activity of a protein kinase specific to this cell line. This protein kinase activity shares some of the distinctive properties of the protein kinases of transforming viruses, i.e., specificity for tyrosine residues, association with membranous and/or cytoskeletal structures, and inhibition by a synthetic peptide derived from the phosphorylation site of pp60src. In view of the absence of a transforming gene in MoMuLV , it is likely that the high level of protein kinase detected in the LSTRA cell line arises from the expression of a cellular gene.     

Vesicular stomatitis virus produced from infected LSTRA lymphoma cells bear tyrosine protein kinase activity (p56)

Murine LSTRA lymphoma cells contain a very active tyrosine protein kinase of 56 kDa (p56) which is not related to any of the other known tyrosine kinases. In the past the purification and characterization of the p56 have been hampered because of the low amount of this protein in LSTRA membranes. In this study, we have utilized a different approach for purification which consisted of trapping the protein in the membrane of vesicular stomatitis virus. Incubation of the virions with [gamma-32P]ATP resulted in the phosphorylation of p56 on tyrosine residues. Moreover, the phosphopeptide digest profile of vesicular stomatitis virus-p56 was identical to that observed with authentic LSTRA-p56. The p56 from such virions could be resolved from other proteins by two-dimensional gels, and furthermore, such virions have been used to prepare several antisera directed against the p56.     

Tumor promoters cause changes in the state of phosphorylation and apparent molecular weight of a tyrosine protein kinase in T lymphocytes

The T cell lymphoma LSTRA contains an elevated level of a tyrosine protein kinase of molecular weight of 56,000 (pp56Tcell) that is present in normal T lymphocytes. Treatment of 32P-labeled LSTRA cells with the phorbol ester 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA), followed by immunoprecipitation of pp56Tcell, revealed that PMA causes complex changes in the state of phosphorylation of pp56Tcell, and the appearance of several new forms of pp56Tcell with higher apparent molecular weights on sodium dodecyl sulfate-polyacrylamide gels. The 32P-labeled pp56Tcell from untreated LSTRA cells contains phosphotyrosine and phosphoserine in a ratio of 2:1. After treatment of LSTRA cells with PMA, the form of pp56Tcell that runs with a molecular weight of 56,000 has approximately equal amounts of phosphotyrosine and phosphoserine, while the higher molecular weight forms of pp56Tcell seen after PMA have 3-4 times more phosphoserine than phosphotyrosine. The induction by PMA of higher molecular weight forms of pp56Tcell could also be demonstrated in preparations of normal human T lymphocytes. The changes in the state of phosphorylation of pp56Tcell after treatment of cells with PMA are consistent with the possibility that pp56Tcell is an in vivo substrate for protein kinase C and provide documentation for a linkage between a mitogenic agent and pp56Tcell.     

Isolation of a human cDNA encoding a 25 kDa FK-506 and rapamycin binding protein.

Recently, the nearly complete peptide sequence of a 25 kDa rapamycin and FK-506 binding protein that had been isolated from calf thymus, brain, and spleen was reported (1). Based upon the amino acid sequence of this bovine protein, bFKBP25, we have isolated from a JURKAT cDNA library the cDNA encoding the human homolog, hFKBP25. Translation of the open reading frame contained within this cDNA clone yields a sequence that, in its C-terminal half, is 41% identical to the major human FK-506 binding protein, hFKBP12, and 43% identical to hFKBP13. The N-terminal half of hFKBP25 is unrelated to any known protein.     

Demonstration that LSTRA cells have an elevated level of proteins phosphorylated on tyrosine residues

The LSTRA cell line has been shown to have an exceptionally high level of a tyrosine protein kinase (pp56lck). We now report that LSTRA cells also have a much higher level of proteins phosphorylated on tyrosine residues in comparison to several other cell lines with normal levels of pp56lck. The level of phosphotyrosine-containing proteins in LSTRA cells was comparable to that seen in K562 cells, a cell line known to have a constitutively active tyrosine protein kinase. These results provide evidence that LSTRA cells have an elevated level of in vivo tyrosine protein kinase activity, probably due to the overexpression and activation of pp56lck.     

Primary structure of the human, membrane-associated Ca2+-binding protein p68 a novel member of a protein family.

cDNA clones encoding human 'p68', a membrane-associated Ca2+-binding protein, were isolated from a lambda gt11 expression library of the human T-leukaemia cell line J6, by using a rabbit antiserum against denatured purified lymphocyte p68, and from a liver cDNA library by using 32P-labelled p68 cDNA fragments. The amino acid sequence of p68, deduced from the sequences of overlapping cDNA clones, is described. The results show that p68 is closely related to a family of proteins which includes intracellular substrates of the EGF receptor and pp60src tyrosine kinases. The p68 amino acid sequence is internally repetitive, being constructed from eight repeats of varying lengths, each of which contains a highly conserved sequence. Multiple copies of the latter sequence are also present in the related proteins p36, lipocortin I and protein II. We discuss how the common structural features of these proteins may reflect common functions and, furthermore, how the eight repeat structure of p68 may have evolved. The sequences of independent cDNAs suggest that alternatively-spliced mRNAs could encode different p68 protein species. This suggestion is consistent with the observation that purified p68 migrates as a closely-spaced doublet when analysed by SDS-PAGE.