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Geldanamycin belongs to the family of benzoquinoid ansamycin tyrosine-kinase inhibitors. We have examined its effects on Her-2/neu kinase activity, protein expression level, and proliferation of Her-2+ malignant cells. In SK-BR-3 breast-cancer cells, short-time treatment with geldanamycin completely abrogated gp30-ligand-induced activation of Her-2 without a change of receptor-expression level. Longer treatment of intact cells with geldanamycin induced decreased steady-state Her-2 autophosphorylation activity, which correlated with reduction of Her-2 protein expression and phosphotyrosine content of several proteins. The decrease was time- and dose-dependent, starting after 1 hr at 100 nM concentration and reaching completion by 24 hr. The reduction of the Her-2 protein level probably resulted from increased degradation, since the Her-2 mRNA level remained constant. Geldanamycin effects were not specific for Her-2, since the non-receptor tyrosine-kinase fyn was inhibited equally. In contrast to these results, protein-kinase-C activity was not affected. In 3 other malignant cell lines expressing different amounts of Her-2 (SK-BR-3 > SK-OV-3 > OVCAR3 > MCF7), geldanamycin also effectively reduced Her-2-kinase activity proportionally to the decrease of protein expression. In contrast, in a [3H]-thymidine-uptake assay, cell growth was meaningfully inhibited by geldanamycin at nanomolar concentrations only in SK-BR-3 (IC50 2 nM) and MCF7 (IC50 20 nM), while OVCAR3 was only moderately sensitive (IC50 2 microM) and SK-OV-3 was clearly resistant to geldanamycin. In direct comparison with herbimycin A, another benzoquinoid ansamycin that has been more thoroughly characterized, the biologic effects of geldanamycin were more pronounced.

Infection with Herpesvirus saimiri, a tumor virus of non-human primates, transformed human CD4+ T cell clones to permanent interleukin (IL)-2-dependent growth without need for restimulation with antigen and accessory cells. The IL-2-dependent proliferation of these cells was dramatically inhibited by soluble anti-CD4 whole antibodies, F(ab')2 and Fab fragments, and also by gp 120 of human immunodeficiency virus. The inhibition was not due to cell death and could be overcome by high concentrations of exogenous IL-2. Cell surface expression of CD4, and to a lesser degree the density of the IL-2 receptor alpha chain, were reduced upon anti-CD4 treatment. After long lasting (> 12 h) incubation with anti-CD4, abundance and activity of CD4-bound p56lck were diminished while the free fraction of p56lck remained unchanged. Since IL-2 binding to its receptor activated only the CD4-bound fraction of p56lck, the IL-2-induced p56lck activity was diminished after long-term CD4 ligation. Taken together, our results suggest a cross talk between CD4- and IL-2 receptor-mediated signaling via p56lck.

CD45 is a tyrosine phosphatase expressed in all hematopoietic cells which is important for signal transduction through the T cell antigen receptor (TCR). Studies using CD45-deficient cells have revealed that Lck, a tyrosine kinase thought to be essential for TCR signaling, is hyperphosphorylated on Y505 in the absence of CD45. This site of tyrosine phosphorylation negatively regulates the function of the Src family of kinases. Here we provide evidence that CD45 can modulate the binding of the Lck to an 11 amino acid tyrosine phosphorylated peptide containing the carboxy-terminus of Lck (lckP). Significantly, CD45 did not influence the binding of Fyn, PLC gamma 1, GAP and Vav to the same phosphopeptide. Lck protein which bound the peptide was dephosphorylated on Y505 and consisted of only 5-10% of the total cellular Lck. Interestingly, there was a marked increase in binding 15-30 min after CD4 or TCR cross-linking. Taken together, our data suggest that CD45 specifically modulates the conformation of Lck in a manner consistent with the intramolecular model of regulation of Src-like kinases.

The tyrosine protein kinases are enzymes that are important in cellular signal transduction. Therefore, inhibition of TPKs provides an important means of investigating and potentially controlling many signaling pathways. The first basic protocol in this unit describes an assay of the inhibitory effects of TPK inhibitors in vitro on a specific TPK that has been immune-precipitated from cell lysates. An assay of the effects of several TPK inhibitors on TPKs in vivo in activated cells is also provided. Although the example used here is a nonreceptor TPK, these protocols can be used to assay the effects of inhibitors on receptor TPKs as well.

We have examined the effect of polyoma virus infection of primary mouse embryo cells on the tyrosyl kinase activity associated with the cellular src gene product, pp60c-src. The results of our studies demonstrate that infection of mouse cells with wild-type polyoma virus or viral mutants capable of transforming rodent cells in culture and inducing tumors in animals results in the stimulation of pp60c-src tyrosyl kinase activity. The level of pp60c-src kinase stimulation in infected cells was found to be proportional to both the oncogenic potential of the virus strain used for infection and the characteristic phenotype of rodent cells transformed by the various strains of polyoma virus. Stimulation of pp60c-src kinase activity was not observed in mouse cells infected with transformation-defective strains of polyoma virus. In examining the kinetics of pp60c-src kinase stimulation in mouse cells at various times following wild-type polyoma virus infection, we found that the level of pp60c-src kinase activity correlated directly with the synthesis of polyoma virus-encoded tumor antigens. By comparing wild-type polyoma virus with other viral mutants in these experiments, we conclude that the stimulation of pp60c-src kinase activity in mouse cells following polyoma virus infection is associated with the synthesis of middle tumor antigen.

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.

The C-SRC, C-YES, and FYN genes encode three closely related tyrosine protein kinases that are expressed in human neural tissues. A unique form of the C-SRC gene has been demonstrated to be expressed in avian and murine brain tissues as the result of alternative splicing between the third and fourth exons. This neuronal-specific splicing event adds to the C-SRC mRNA an 18 base pair exon capable of encoding the same six amino acids in both avian and murine neural tissues. The C-YES and FYN genes share with C-SRC similar exon-intron boundaries and a high degree of amino acid sequence homology in the 3/4 exon coding region. However, potential alternative splicing of the C-YES and FYN genes in this region has not been previously investigated. In this study we have compared the expression of C-SRC, C-YES, and FYN RNAs in human lung, liver, brain, and placenta tissues and prepared cDNA clones spanning exons 3 and 4 for each of these genes from the different tissues. Sequence analysis of these cDNA clones revealed that the splicing patterns for the FYN and C-YES genes were the same among the various tissues, whereas C-SRC cDNAs isolated from brain contained 18 additional bases with the capacity to code for the same six amino acids present in the neural-specific forms of avian and murine pp60c-src.