Phosphatidylinositol turnover and transformation of cells by Abelson murine leukaemia virus.

The transforming protein of the Abelson murine leukaemia virus encodes a protein-tyrosine kinase. Previously, we have shown that in Abelson-transformed cells, the Abelson kinase regulates the phosphoserine content of ribosomal protein S6. Phorbol 12-myristate 13-acetate (TPA), which activates protein kinase C, induces the phosphorylation of S6 at the same five phosphopeptides as found in S6 isolated from Abelson-transformed cells. We have investigated three models whereby the Abelson kinase might regulate S6 phosphorylation via the activation of protein kinase C. First, the Abelson kinase could phosphorylate protein kinase C on tyrosine. However, we do not detect significant amounts of phosphotyrosine in protein kinase C in vivo. Second, it has been suggested that protein-tyrosine kinases might phosphorylate phosphatidylinositol. This could increase the intracellular levels of diacylglycerol and thereby activate protein kinase C. Our data strongly suggest that direct phosphorylation of phosphatidylinositol by the Abelson protein-tyrosine kinase has no physiological role. Third, an indirect activation of protein kinase C may occur via an increase in the rate of phosphoinositide breakdown. We have found that phosphoinositide breakdown appears to be constitutively activated in Abelson-transformed cells. The implications of these observations are discussed with regard to S6 phosphorylation and the mechanism of Abelson-induced transformation.     

Only site-directed antibodies reactive with the highly conserved src-homologous region of the v-abl protein neutralize kinase activity

Antisera specific for six regions of the v- abl protein were used to serologically characterize the Abelson murine leukemia virus tyrosine kinase. Chemically synthesized peptides corresponding to the predicted v- abl protein sequence and larger regions of the v- abl protein expressed as fusion proteins in bacteria were used as immunogens. The specificity of each antiserum was confirmed by immunoprecipitation analysis with defined deletion mutants of Abelson murine leukemia virus. Several of these v- abl -specific antisera display much higher titers and avidities than serum harvested from mice bearing Abelson murine leukemia virus-induced tumors, previously the only source of anti- abl -specific serum. Two antisera were found to block the in vitro autophosphorylation of the v- abl protein as well as its ability to phosphorylate a peptide substrate. Examination of the sites against which the kinase-blocking antisera were prepared revealed that both are in close proximity to the in vivo sites of tyrosine phosphorylation, which fall within the region of high homology with v-src and other tyrosine kinases. Antisera directed against other regions of v- abl did not inhibit kinase activity.     

Phosphorylation and activation of p40 tyrosine kinase by casein kinase-1

Because examination of regulatory trans-phosphorylations can help elucidate the cellular functions of tyrosyl protein kinases, we have investigated the effects of phosphorylation by casein kinase-1 on the activity of the p40 tyrosyl protein kinase. We find that casein kinase-1 can phosphorylate the p40 tyrosyl kinase on serine and threonine residues, in part on a unique tryptic peptide. The phosphorylation induces a substantial increase in the tyrosyl protein kinase activity of p40, in contrast to most instances in which serine/threonine phosphorylation inhibits activity of tyrosyl protein kinases. These findings raise the possibility that p40 might be part of a protein phosphorylation network in which casein kinase-1 participates.     

Identification of p42 Mitogen-Activated Protein Kinase as a Tyrosine Kinase Substrate Activated by Maximal Electroconvulsive Shock in Hippocampus

Abstract: Recent studies have demonstrated that administration of an electroconvulsive shock produces a rapid and transient increase in tyrosyl phosphorylation of a ∼40-kDa protein in rat brain. Initial characterization of this protein's chromatographic properties indicated that it might be a member of a recently identified family of kinases, referred to as mitogen-activated protein (MAP) kinases, that are activated by tyrosyl phosphorylation. In the present study, we have used MAP kinase antisera to assess the identity of this protein. We have found that the ∼40-kDa phosphotyrosine-containing protein comigrates with p42 MAP kinase (p42^mapk) and not with two other 44-kDa MAP kinase family members detected by these antisera. Western blots of proteins immunoprecipitated with MAP kinase antibodies confirm that p42^mapk displays increased tyrosyl phosphorylation after an electroconvulsive stimulus. Chromatographic separation of hippocampal extracts indicates that MAP kinase activity elutes in parallel with p42^mapk. Accordingly, these studies identify p42^mapk as a tyrosyl kinase substrate that is activated by this stimulus and suggest that this form of MAP kinase may be selectively regulated by neuronal stimulation.     

Inhibition of tyrosine protein kinases by the antineoplastic agent adriamycin

Adriamycin, a lipid-interacting anti-cancer agent, was found to inhibit the phosphorylation of polyGlu/Tyr (4:1) by tyrosine protein kinases either from spleen or expressed by the oncogene of Abelson murine leukemia virus. The dose dependent inhibition by adriamycin is accounted for by competition for the ATP binding site, but it is also deeply influenced by the nature and concentration of the phosphorylatable substrate, suggesting multiple interactions with the enzyme. The phosphorylation at tyrosine residues of cytosolic proteins from cells transformed by Abelson leukemia virus and the autophosphorylation of tyrosine protein kinases are also inhibited by adriamycin. Unlike tyrosine protein kinases most serine/threonine specific protein kinases, with the notable exception of protein kinase-C, appear to be relatively insensitive to adriamycin.     

Specific inhibition of tyrosine kinase activity by an antibody to the v-ros oncogene product.

Antibodies present in two peritoneal exudates of rats bearing abdominal tumors induced by UR2-transformed rat cells were characterized. The ability to immunoprecipitate p68gag-ros and to inhibit the protein and phospholipid kinase activities of this protein was investigated. One of the exudates specifically inhibited tyrosyl phosphorylation by p68gag-ros but not the activity of other known tyrosyl kinases, such as p150gag-fps of UR1 avian sarcoma virus, p60src, and the insulin receptor. It precipitated p68gag-ros but not Pr76 or other gag-related proteins from UR2-infected cells. Phosphorylation of phosphatidylinositol was not affected by this exudate, suggesting that this activity is not intrinsic to p68gag-ros. Another exudate precipitated p68gag-ros but not gag-related proteins from UR2-infected cells or p140gag-fps from Fujinami sarcoma virus-infected cells. These results demonstrated that the antibodies in these exudates recognized epitopes present in the ros portion of the fused protein p68gag-ros, but only one of the two exudates inhibited the intrinsic tyrosyl kinase of p68gag-ros.     

Characterization and use of monoclonal antibodies for isolation of phosphotyrosyl proteins from retrovirus-transformed cells and growth factor-stimulated cells.

Protein kinases that phosphorylate the hydroxyl group of tyrosine residues of proteins have been implicated in cell transformation by some retroviruses and in regulation of normal cell growth by some polypeptide growth factors. To facilitate the identification of tyrosine kinase substrates, we developed monoclonal antibodies to the hapten azobenzylphosphonate. One of these antibodies, MA-2G8, proved to be especially attractive in that it bound a derivative of aminophenylphosphate, a close phosphotyrosine analog, with higher affinity than it bound the corresponding derivative of aminobenzylphosphonate; however, its affinity for phosphoserine was negligible. In this paper we describe the optimal conditions for using this antibody to isolate phosphotyrosine proteins, emphasizing particularly that its interaction with phosphotyrosyl proteins is sensitive to ionic detergents and to antibody density on the immunosorbent matrix. The antibody also bound ATP citrate lyase; this enzyme lacks phosphotyrosine but contains phosphohistidine, which is similar structurally to phosphotyrosine. By attaching the antibody at high density to Sepharose beads and omitting ionic detergents from the buffers, it was possible by microbatch immunoadsorption (followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to isolate the 120,000-dalton transforming protein and several other phosphotyrosyl proteins from cells transformed by Abelson murine leukemia virus. Under the same conditions, phosphotyrosyl proteins were also isolated from human epidermal carcinoma cells (A431) that had been stimulated with epidermal growth factor; most prominent among these proteins was the 170,000-dalton receptor for epidermal growth factor.     

In vitro association and phosphorylation of polyoma virus middle T antigen by cellular tyrosyl kinase activity

We have observed increased phosphorylation of tyrosine residues on the polyoma virus middle tumor antigen (MTAg) in in vitro kinase assays of the immune complexes immunoprecipitated from lysates of polyoma virus-infected mouse embryo cells to which increasing amounts of uninfected mouse embryo cell lysate had been added. The components from uninfected mouse cells responsible for increased MTAg phosphorylation were localized by subcellular fractionation to the plasma membrane and found to be sensitive to protease digestion, N-ethylmaleimide, and 5'-p-fluorosulfonylbenzoyladenosine inactivation. The majority of the membrane-associated activity responsible for the increased MTAg phosphorylation in these assays could be cleared from lysates of uninfected mouse cell lysates by centrifugation after reaction with Sepharose-bound monoclonal antibodies which recognize pp60c-src. These results suggest that MTAg can associate with cellular tyrosyl kinases in vitro and be phosphorylated by these enzymes in immune-complex kinase assays. The identity of at least one of these cellular tryosyl kinases which can associate with MTAg in vitro is likely to be pp60c-src.     

Tyrosyl kinases acquired from anchorage-independent cells by a membrane-enveloped virus

Tyrosyl kinase activity in vesicular stomatitis virus (VSV) acquired from host cells that differ in morphology was investigated. VSV grown in baby hamster kidney (BHK) cells with rounded morphology and a high efficiency of colony formation in soft agar (Rous sarcoma virus [RSV]- transformed and suspension BHK cells) was compared with VSV grown in BHK cells with a flattened morphology and lower efficiency of colony formation in soft agar (RSV-infected revertant and control BHK cells). Tyrosyl kinase activity measured with the substrates angiotensin II peptide or casein was found at 7-10-fold higher levels in virus released from the anchorage-independent BHK cells. Most of the VSV- associated tyrosyl kinases acquired from the RSV-transformed BHK cells reacted with antiserum to pp60src, whereas the activity acquired from the suspension BHK cells was unaffected by anti-src serum. The overall levels of tyrosyl kinase in subcellular fractions of the host BHK cells were also measured. Like the VSV released from them, the RSV- transformed cell extracts contained high levels. The suspension cells, however, contained the same low levels of tyrosyl kinase as was found in the control BHK cell extracts. Therefore, tyrosyl kinase was concentrated and acquired by VSV from the anchorage-independent suspension BHK cells. VSV-associated protein kinases acquired from other cell types followed a similar pattern. Tyrosyl kinase levels were high in VSV released from suspension cultures (Chinese hamster ovary and HeLa) and from virally transformed cells (Kirsten murine sarcoma virus-transformed rat kidney cells) and low in VSV released from an anchorage-dependent primary cell culture (chick embryo fibroblasts).     

Association of ERK2 mitogen-activated protein kinase with human immunodeficiency virus particles.

Here we report the presence of a protein kinase activity associated with human immunodeficiency virus type 1 (HIV-1) particles. We observed phosphorylation of five major proteins by the endogenous protein kinase activity. Phosphoamino acid analysis revealed phosphorylated serine and threonine residues. In addition, we observed autophosphorylation of two proteins in the presence of gamma-ATP in an in-gel phosphorylation assay. These two proteins are not linked by a disulfide bond, suggesting that two different protein kinases are associated with HIV-1 virions. Our results indicate the presence of ERK2 mitogen-activated protein kinase and of a 53,000-molecular-weight protein kinase associated with virions. Moreover, the use of different HIV strains derived from T cells and promonocytic cells, as well as the use of human T-cell leukemia virus type 1 particles, demonstrates that ERK2 is strongly associated with retrovirus particles in a cell-independent manner. Exogenous substrates, such as histone proteins, and a viral substrate, such as Gag protein, are phosphorylated by virus-associated protein kinases.     

Abelson murine leukemia virus mutants deficient in kinase activity and lymphoid cell transformation.

Abelson murine leukemia virus transforms both lymphoid cells and fibroblasts in vitro and induces a unique type of thymus-dependent lymphoma in vivo. Four fibroblast-transforming strains of Abelson murine leukemia virus were identified, based on the sizes of the Abelson murine leukemia virus-specific phosphoproteins produced by these isolates. Two of these strains, the standard P120- and the P160-producing viruses, transformed lymphoid cells efficiently in vitro and induced Abelson disease in vivo. Two other strains, which synthesized small Abelson murine leukemia virus-specific proteins with molecular weights of 90,000 (P90) and 100,000 (P100), transformed lymphoid cells very poorly both in vitro and in vivo. The reduced oncogenic potentials of these isolates were correlated with a high level of synthesis of fairly unstable P90 and P100. In addition, neither P90 nor P100 functional efficiently in protein kinase assays. The correlation of abnormal metabolism and deficient protein kinase activity with the reduced oncogenic potentials of these virus strains supported a direct role for these proteins and the kinase activity in transformation. Furthermore, these results suggested that the requirements for lymphoid cell transformation and fibroblast transformation are different.     

Some lymphoid cell lines transformed by Abelson murine leukemia virus lack a major 36,000-dalton tyrosine protein kinase substrate.

Fibroblasts transformed by Abelson murine leukemia virus differ from normal fibroblasts in that they contain several cellular proteins, including one of 29 and one of 36 kilodaltons, which are phosphorylated at tyrosine residues. Since it has been shown before that these proteins also become phosphorylated at tyrosine after transformation of fibroblasts by a number of other retroviruses, their phosphorylation may play an important role in the transformation of these cells. In contrast, the 36-kilodalton phosphoprotein was not detectable in three of the four lines of Abelson virus-transformed B lymphoma cell lines studied here. These three cell lines, RAW307.1.1, 18-48, and 18-81, and a B lymphoma induced by mineral oil, WEHI 279, were all found to lack both the phosphorylated and unphosphorylated forms of the 36-kilodalton protein. It thus appears that expression of this major cell protein is not essential for the survival of B lymphoma cells in culture and that the phosphorylation of the 36-kilodalton protein at tyrosine is not essential for transformation of pre-B lymphocytes by Abelson virus.     

Identification of the regulatory phosphorylation sites in pp42/mitogen-activated protein kinase (MAP kinase).

Mitogen-activated protein kinase (MAP kinase) is a 42 kd serine/threonine protein kinase whose enzymatic activity requires phosphorylation of both tyrosyl and threonyl residues. As a step in elucidating the mechanism(s) for activation of this enzyme, we have determined the sites of regulatory phosphorylation. Following proteolytic digestion of 32P-labeled pp42/MAP kinase with trypsin, only a single phosphopeptide was detected by two-dimensional peptide mapping, and this peptide contained both phosphotyrosine and phosphothreonine. The amino acid sequence of the peptide, including the phosphorylation sites, was determined using a combination of Fourier transform mass spectrometry and collision-activated dissociation tandem mass spectrometry with electrospray ionization. The sequence for the pp42/MAP kinase tryptic phosphopeptide is similar (but not identical) to a sequence present in the ERK1- and KSS1-encoded kinases. The two phosphorylation sites are separated by only a single residue. The regulation of activity by dual phosphorylations at closely spaced threonyl and tyrosyl residues has a functional correlate in p34cdc2, and may be characteristic of a family of protein kinases regulating cell cycle transitions.     

STI-571: an anticancer protein-tyrosine kinase inhibitor

STI-571 (imatinib, Gleevec, Glivec, CGP 57148) is an inhibitor of the Abl group of protein-tyrosine kinases. One of these enzymes, the Bcr-Abl oncoprotein, results from the fusion of the BCR and ABL genes that result from the reciprocal chromosomal translocation that forms the Philadelphia chromosome. The Philadelphia chromosome occurs in 95% of people with chronic myeloid leukemia. ABL is the cellular homologue of the oncogene found in murine Abelson leukemia virus, and BCR refers to breakpoint cluster region. The Bcr-Abl oncoprotein exhibits elevated protein-tyrosine kinase activity, which is strongly implicated in the mechanism of development of chronic myeloid leukemia. STI-571 is effective in the treatment of the stable phase of chronic myeloid leukemia. The c-Abl protein kinase domain exists in an active and inactive conformation. STI-571 binds only to the inactive state of the enzyme as shown by X-ray crystallography. The drug binds to a portion of the ATP-binding site and extends from there into adjacent hydrophobic regions. STI-571 is a competitive inhibitor of Abl kinase with respect to ATP. Resistance to STI-571 is often the result of mutations in residues of the Bcr-Abl kinase that ordinarily bind to the drug. Inhibition of target protein kinases represents an emerging therapeutic strategy for the treatment of cancer.     

Purification and characterization of a protein kinase from Xenopus eggs highly specific for ribosomal protein S6

In Xenopus oocytes ribosomal protein S6 becomes phosphorylated on serine residues in response to hormones or growth factors and following microinjection of the tyrosine-specific protein kinases associated with Rous sarcoma virus or Abelson murine leukemia virus. To begin characterization of the enzymes responsible for S6 phosphorylation in this system, we have undertaken the purification of S6 protein kinases from unfertilized Xenopus eggs. DEAE-Sephacel chromatography of crude extracts revealed two peaks of S6 kinase activity, and the peak eluting at 160 mM NaCl was chosen for further purification. Successive chromatography on Mono S, Sephacryl S-200, Mono Q, and heparin-Sepharose resulted in purification of the enzyme to a single protein migrating at Mr = 92,000 on polyacrylamide gels. The final preparation was purified about 500-fold from the DEAE-Sephacel peak with a recovery of 10%. Apparent Km values of the enzyme for ATP and 40 S subunits were 28 and 5 microM, respectively, and the specific activity with 330 microM ATP and 5.6 microM 40 S subunits was 300 nmol/min/mg. The enzyme was inhibited by beta-glycerophosphate, sodium fluoride, potassium phosphate, ADP, heparin, quercetin, and spermine. The availability of a purified S6 protein kinase should facilitate elucidation of the molecular mechanism of S6 phosphorylation during growth stimulation.     

Characterization of the novel protein kinase activity present in the R1 subunit of herpes simplex virus ribonucleotide reductase.

We have compared the protein kinase activities of the R1 subunits from herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) ribonucleotide reductase following expression in Escherichia coli. Autophosphorylation activity was observed when kinase assays were performed with immunoprecipitated R1 or proteins purified to homogeneity, and the activity was stimulated by the basic protein protamine. Transphosphorylation of histones or calmodulin by purified or immunoprecipitated HSV-1 and HSV-2 R1 was not observed, and our results suggest that the activities of these two proteins are similar. We further characterized the protein kinase activity of HSV-1 R1 by producing insertion and deletion mutants constructed with a plasmid expressing R1 amino acids 1 to 449. C-terminal deletion analysis identified the catalytic core of the enzyme as comprising residues 1 to 292, and this polypeptide will be useful for structural determinations by X-ray crystallography. Insertion of a 4-amino-acid sequence at sites within the protein kinase domain identified regions essential for activity; insertions at residues 22 and 112 completely inactivated activity, and an insertion at residue 136 reduced activity sixfold. Similar insertions at residues 257, 262, 292, and 343 had no effect on activity. The ATP analog 5'-fluorosulfonylbenzoyladenosine, which covalently modifies conventional eukaryotic kinases at an essential lysine residue within the active site, did label HSV R1, but this labelling occurred outside the N-terminal domain. These data indicate that the HSV R1 kinase is novel and distinct from other eukaryotic protein kinases.     

Four different classes of retroviruses induce phosphorylation of tyrosines present in similar cellular proteins.

Chicken embryo cells transformed by the related avian sarcoma viruses PRC II and Fujinami sarcoma virus, or by the unrelated virus Y73, contain three phosphoproteins not observed in untransformed cells and increased levels of up to four other phosphoproteins. These same phosphoproteins are present in increased levels in cells transformed by Rous sarcoma virus, a virus which is apparently unrelated to the three aforementioned viruses. In all cases, the phosphoproteins contain phosphotyrosine and thus may be substrates for the tyrosine-specific protein kinases encoded by these viruses. In one case, the site(s) of tyrosine phosphorylation within the protein is the same for all four viruses. A homologous protein is also phosphorylated, at the same major site, in mouse 3T3 cells transformed by Rous sarcoma virus or by the further unrelated virus Abelson murine leukemia virus. A second phosphotyrosine-containing protein has been detected in both Rous sarcoma virus and Abelson murine leukemia virus-transformed 3T3 cells, but was absent from normal 3T3 cells and 3T3 cells transformed by various other viruses. We conclude that representatives of four apparently unrelated classes of transforming retroviruses all induce the phosphorylation of tyrosines present in the same set of cellular proteins.     

Phosphorylation of rabbit and human erythrocyte membranes by soluble adenosine 3':5'-monophosphate-dependent and -independent protein kinases.

Previous reports from this laboratory and others have established that both the rabbit and human erythrocyte membranes contain multiple protein kinase and phosphate acceptor activities. We now report that these membranes also contain phosphoryl acceptor sites for the soluble cyclic AMP-dependent and -independent protein kinases from rabbit erythrocytes. The rabbit erythrocyte membrane, which does not contain a cyclic AMP-dependent protein kinase, has at least four polypeptides (Bands 2.1, 2.3, 4.5, and 4.8) which are phosphorylated in the presence of the soluble cyclic AMP-dependent protein kinases I, IIa, and IIb isolated from rabbit erythrocyte lysates. The resulting phosphoprotein profile is very similar to that obtained for the cyclic AMP-mediated autophosphorylation of human erythrocyte membranes. The activities of the soluble cyclic AMP-dependent protein kinases toward the membranes have been studied at several pH values. Although the substrate specificity of the three kinases is similar, polypeptide 2.3 appears to be phosphorylated to a greater extent by kinase IIa than by I or IIb. This occurs at all pH values studied. Also apparent is that the pH profile for membrane phosphorylation is different from that of histone phosphorylation. The phosphorylation of membrane proteins can also be catalyzed by the soluble erythrocyte casein kinases. These enzymes are not regulated by cyclic nucleotides and can use either ATP or GTP as their phosphoryl donor. Polypeptides 2.1, 2.9, 4.1, 4.5, 4.8, and 5 of both human and rabbit erythrocyte membranes are phosphorylated in the presence of GTP and the casein kinases. This reaction is optimal at pH 7.5. Experiments were performed to determine whether the phosphorylation of the membranes by the soluble and membrane-bound kinases is additive or exclusive. Our results indicate that after maximal autophosphorylation of the erythrocyte membranes, phosphoryl acceptor sites are available to the soluble cyclic AMP-dependent and -independent protein kinases. Furthermore, after maximal phosphorylation of the membranes with one type of soluble kinase, further 32P incorporation can occur as a result of exposure to the other type of soluble kinase.     

A noncatalytic domain conserved among cytoplasmic protein-tyrosine kinases modifies the kinase function and transforming activity of Fujinami sarcoma virus P130gag-fps.

Proteins encoded by oncogenes such as v-fps/fes, v-src, v-yes, v-abl, and v-fgr are cytoplasmic protein tyrosine kinases which, unlike transmembrane receptors, are localized to the inside of the cell. These proteins possess two contiguous regions of sequence identity: a C-terminal catalytic domain of 260 residues with homology to other tyrosine-specific and serine-threonine-specific protein kinases, and a unique domain of approximately 100 residues which is located N terminal to the kinase region and is absent from kinases that span the plasma membrane. In-frame linker insertion mutations in Fujinami avian sarcoma virus which introduced dipeptide insertions into the most stringently conserved segment of this N-terminal domain in P130gag-fps impaired the ability of Fujinami avian sarcoma virus to transform rat-2 cells. The P130gag-fps proteins encoded by these transformation-defective mutants were deficient in protein-tyrosine kinase activity in rat cells. However v-fps polypeptides derived from the mutant Fujinami avian sarcoma virus genomes and expressed in Escherichia coli as trpE-v-fps fusion proteins displayed essentially wild-type enzymatic activity, even though they contained the mutated sites. Deletion of the N-terminal domain from wild-type and mutant v-fps bacterial proteins had little effect on autophosphorylating activity. The conserved N-terminal domain of P130gag-fps is therefore not required for catalytic activity, but can profoundly influence the adjacent kinase region. The presence of this noncatalytic domain in all known cytoplasmic tyrosine kinases of higher and lower eucaryotes argues for an important biological function. The relative inactivity of the mutant proteins in rat-2 cells compared with bacteria suggests that the noncatalytic domain may direct specific interactions of the enzymatic region with cellular components that regulate or mediate tyrosine kinase function.