A strong protein-tyrosine kinase activity is associated with a baculovirus-expressed chicken tkl gene.

We have previously described a gene named tkl (tyrosine kinase related to lck). It belongs to the src family of protein-tyrosine kinases and among these it has significant homology to the lck gene (lymphoide cell kinase). The tkl gene product may represent the avian homolog of Lck, which is believed to participate in a lymphocyte-specific signal transduction pathway by association with a membrane receptor. To study the biochemical properties of the protein, a nearly complete tkl gene (isolated from a cDNA library from chicken spleen cells) was expressed in a baculovirus system. Approximately 10% of the extracted protein consisted of the soluble 51-kDa Tkl protein (p51tkl) at 40 h post-infection. This protein was found to be phosphorylated on tyrosine and serine residues at a ratio of 5:1. As expected, glycosylation or myristoylation could not be detected. Immunocomplex kinase assays indicated strong autophosphorylation of p51tkl at tyrosine residues and phosphorylation of exogenous substrates such as D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histones H2b and H4, and casein. This protein-tyrosine kinase activity also exhibited a marked preference for Mn2+ compared to Mg2+. The high level expression of enzymatically active Tkl should provide an excellent tool to further study the biological functions of this class of enzymes.     

Identification of a 42-kilodalton phosphotyrosyl protein as a serine(threonine) protein kinase by renaturation.

We have surveyed fibroblast lysates for protein kinases that might be involved in mitogenesis. The assay we have used exploits the ability of blotted, sodium dodecyl sulfate-denatured proteins to regain enzymatic activity after guanidine treatment. About 20 electrophoretically distinct protein kinases could be detected by this method in lysates from NIH 3T3 cells. One of the kinases, a 42-kilodalton serine(threonine) kinase (PK42), was found to possess two- to fourfold-higher in vitro activity when isolated from serum-stimulated cells than when isolated from serum-starved cells. This kinase comigrated on sodium dodecyl sulfate-gels with a protein (p42) whose phosphotyrosine content increased in response to serum stimulation. The time courses of p42 tyrosine phosphorylation and PK42 activation were similar, reaching maximal levels within 10 min and returning to basal levels within 5 h. Both p42 tyrosine phosphorylation and PK42 activation were stimulated by low concentrations of phorbol esters, and the responses of p42 and PK42 to TPA were abolished by chronic 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. Chronic TPA treatment had less effect on serum-induced p42 tyrosine phosphorylation and PK42 activation. PK42 and p42 bound to DEAE-cellulose, and both eluted at a salt concentration of 250 mM. Thus, PK42 and p42 comigrate and cochromatograph, and the kinase activity of PK42 correlates with the tyrosine phosphorylation of p42. These findings suggest that PK42 and p42 are related or identical, that PK42 is activated by tyrosine phosphorylation, and that this tyrosine phosphorylation can be regulated by protein kinase C.     

Identification of the human pim-1 gene product as a 33-kilodalton cytoplasmic protein with tyrosine kinase activity.

The human pim-1 gene was recently identified as a new putative oncogene located on chromosome 6p21, a region showing karyotypic abnormalities in particular leukemias. In the present work we characterized the pim protein product. In vitro translation of positively selected poly(A)+ mRNA indicates that this gene encodes a 33-kilodalton protein. Anti-pim antibodies were raised against a fused TrpE-pim protein induced in a bacterial expression vector. This antibody immunoprecipitated a 33-kilodalton protein from in vivo [35S]methionine-labeled K562 and KCl myelogenous origin cell lines. This protein was localized to the cytoplasm, and in vivo labeling as well as in vitro kinase assay suggests that it is a phosphoprotein with tyrosine kinase activity. This was further confirmed by performing autophosphorylation directly on a p33pim-containing gel band cut out after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results imply that the tyrosine kinase activity of pim can be recovered after boiling the pim-1 protein in sample buffer: a feature not described yet for this class of protein. These results suggest that pim-1 is a new member of the subgroup of oncogenes encoding tyrosine kinases.     

p185, a product of the neu proto-oncogene, is a receptorlike protein associated with tyrosine kinase activity.

The neu oncogene was originally identified in cell lines derived from rat neuroectodermal tumors. neu is related to but distinct from the c-erbB gene, which encodes the epidermal growth factor (EGF) receptor. neu encodes a protein, designated p185, that is serologically related to the EGF receptor. Identification of the normal homolog of p185 encoded by the neu proto-oncogene enabled us to compare the product of the neu proto-oncogene with the mutated version specified by the neu oncogene and with the EGF receptor. The normal form of p185 was structurally similar to its transforming counterpart, indicating that activation of the neu oncogene did not cause major structural alterations in the gene product. Both normal and transforming forms of p185 were associated with tyrosine kinase activity, supporting the idea that normal p185 functions as a growth factor receptor. p185 differed both structurally and functionally from the EGF receptor. p185 and the EGF receptor had distinct electrophoretic mobilities when synthesized under normal culture conditions or in the presence of tunicamycin. EGF did not stimulate increased turnover of p185 and did not bind quantitatively to p185. A number of other growth factors failed to stimulate degradation of p185 or tyrosine phosphorylation of p185 and are therefore unlikely to be ligands for p185.     

A 41-kilodalton protein is a potential substrate for the p210bcr-abl protein-tyrosine kinase in chronic myelogenous leukemia cells.

Chronic myelogenous leukemia (CML) is characterized by a translocation involving the c-abl protein-tyrosine kinase gene. A chimeric mRNA is formed containing sequences from a chromosome 22 gene (bcr) at its 5' end and all but the variable exon 1 of c-abl sequence. The product of this mRNA, p210bcr-abl, has constitutively high protein-tyrosine kinase activity. We examined K562 cells and other lines established from CML patients for the presence of phosphotyrosine (P-Tyr)-containing proteins which might be p210bcr-abl substrates. Two-dimensional gel separation of 32P-labeled proteins followed by phosphoamino acid analysis of 25 phosphoproteins, which comprised the major alkali-stable phosphoproteins, indicated that three related proteins of 41 kDa are the most prominent P-Tyr-containing proteins detected by this method. The 41-kDa phosphoproteins are found in two other CML lines that we examined but not in lines of similar lineage isolated from patients with distinct leukemic disease. A protein that comigrates with the major form of pp41 (pp41A) and contains P-Tyr is also found in murine fibroblasts and B-lymphoid cells transformed by Abelson murine leukemia virus, which encodes the v-abl protein, and in platelet-derived growth factor-treated fibroblasts, in which it has been described previously. We analyzed three pairs of Epstein-Barr virus-immortalized B-cell lines from individual CML patients and found that only the lines in which active p210bcr-abl was present contained detectable pp41. We also performed immunoblotting with anti-P-Tyr antibodies on the same CML cell lines and detected at least four other putative substrates of p210bcr-abl, which were undetected with use of the two-dimensional gel technique.     

The vaccinia virus B1R gene product is a serine/threonine protein kinase.

The nucleotide sequence of the vaccinia virus open reading frame B1 predicts a polypeptide with significant sequence similarity to the catalytic domain of known protein kinases. To determine whether the B1R polypeptide is a protein kinase, we have expressed it in bacteria as a fusion with glutathione S-transferase. Affinity-purified preparations of the fusion protein were found to undergo autophosphorylation and also phosphorylated the exogenous substrates casein and histone H1. Mutation of lysine 41 to glutamine within the conserved kinase catalytic domain II abrogated protein kinase activity on all three protein substrates, supporting the notion that the protein kinase activity is inherent to the B1R polypeptide. Casein and histone H1 were phosphorylated on serine and threonine residues. The B1R fusion protein was phosphorylated on a threonine residue(s) by an apparently intramolecular mechanism. The autophosphorylation reaction resulted in phosphorylation of the glutathione S-transferase portion of the fusion and not the protein kinase domain. The protein kinase activity of B1R was specific for ATP as the phosphate donor; GTP was not utilized to a detectable extent. Immunoblotting experiments with anti-B1R antiserum showed that the protein kinase is located in the virion particle. Chromatography of virion extracts resulted in separation of the B1R protein kinase from the bulk of the total protein kinase activity, indicating that multiple protein kinases are present in the virion particle and that B1R is distinct from the previously described vaccinia virus-associated protein kinase.     

Characterization of protein kinase activity associated with the transforming gene product of Fujinami sarcoma virus

Fujinami sarcoma virus (FSV), a newly characterized avian sarcoma virus, produces a protein of 140,000 daltons (p140) in infected cells. p140 is the product of a fused gene consisting of a part of the gag gene of avian retrovirus and FSV-unique sequences which are not related to the src sequences of Rous sarcoma virus. In vivo, p140 was found to be phosphorylated at both serine and tyrosine residues. Immunoprecipitates of p140 with antiserum against gag gene-coded proteins had a cyclic nucleotide-independent protein kinase activity which phosphorylated p140 itself, rabbit IgG of the immune complex and alpha-casein, an externally added soluble protein substrate. The phosphorylation was specific to tyrosine of the substrate proteins. p140 was phosphorylated in vitro at the same two tyrosine residues that were phosphorylated in vivo. The phosphate transferred to tyrosine residues of p140 forms a stable bond: it does not turn over during the kinase reaction, and the 32P-phosphate of p140 labeled in vitro or in vivo is not transferred to alpha-casein. FSV-p140 differs from p60src, the transforming protein of Rous sarcoma virus, in its marked preference of Mn2+ to Mg2+ ions, and in its inability to use GTP instead of ATP as the donor of gamma-phosphate.     

Evidence that the Rous sarcoma virus transforming gene product is associated with glycerol kinase activity

This communication provides biochemical, immunological, and genetic evidence that pp60src, the Rous sarcoma virus transforming gene product, is associated with glycerol kinase activity. Our investigations demonstrated that the compound phosphorylated by pp60src or by glycerol kinase (EC 2.7.1.30) from Candida mycoderma share the same electrophoretic and chromatographic mobilities. The glycerol kinase and protein kinase activities of pp60src were inhibited similarly by preincubation with immune IgG. Both activities were reduced 6-9-fold in pp60src preparations derived by immunoaffinity chromatography from cells which were infected with NY68, a temperature-sensitive transformation mutant of Rous sarcoma virus. The thermolability at 41 degrees C of the glycerol kinase activity of pp60src from the mutant virus-infected cells was greater (t/2 = 1.3 min) than the same activity in pp60src preparations from wild type virus-infected cells (t/2 = 4.8 min).     

The human c-fps/fes gene product expressed ectopically in rat fibroblasts is nontransforming and has restrained protein-tyrosine kinase activity.

A 13-kilobase EcoRI genomic restriction fragment containing the human c-fps/fes proto-oncogene locus was expressed transiently in Cos-1 monkey cells and stably in Rat-2 fibroblasts. In both cases, human c-fps/fes directed synthesis of a 92-kilodalton protein-tyrosine kinase (p92c-fes) indistinguishable from a tyrosine kinase previously identified with anti-fps antiserum which is specifically expressed in human myeloid cells. Transfected Rat-2 cells containing approximately 50-fold more human p92c-fes than is found in human leukemic cells remained morphologically normal and failed to grow in soft agar. Synthesis of p92c-fes in this phenotypically normal line exceeded that of the P130gag-fps oncoprotein in a v-fps-transformed Rat-2 line. Despite this elevated expression, human p92c-fes induced no substantial increase in cellular phosphotyrosine and was not itself phosphorylated on tyrosine. In contrast, p92c-fes immunoprecipitated from these Rat-2 cells or expressed as an enzymatically active fragment in Escherichia coli from a c-fps/fes cDNA catalyzed tyrosine phosphorylation with an activity similar to that of v-fps/fes polypeptides. Thus, p92c-fes is not transforming when ectopically overexpressed in Rat-2 fibroblasts. This lack of transforming activity correlates with a restriction imposed on the kinase activity of the normal c-fps/fes product in vivo which is apparently lifted for v-fps/fes oncoproteins, suggesting that regulatory interactions within the host cell modify fps/fes protein function and normally restrain its oncogenic potential.     

The CDC4 gene product is associated with the yeast nuclear skeleton

The CDC4 gene product of Saccharomyces cerevisiae is required at the late G1/S phase boundary of the cell cycle. In an attempt to better understand the function of CDC4, we performed experiments to localize this protein in the yeast cell. Using antisera, directed against a TrpE-CDC4 fusion protein, to analyze immuno-blots of different subcellular fractions from yeast, we demonstrated that the CDC4 gene product localizes in the nucleus by two different biochemical preparations of the yeast nucleoskeletal proteins. Immunofluorescence microscopy further confirmed its nuclear localization. These data support a model that includes the CDC4 gene product as a component of the yeast nuclear skeleton. The significance of this association in relationship to the biological role of CDC4 is discussed.     

Calcium-independent activation of salicylic acid-induced protein kinase and a 40-kilodalton protein kinase by hyperosmotic stress

Reversible protein phosphorylation/dephosphorylation plays important roles in signaling the plant adaptive responses to salinity/drought stresses. Two protein kinases with molecular masses of 48 and 40 kD are activated in tobacco cells exposed to NaCl. The 48-kD protein kinase was identified as SIPK (salicylic acid-induced protein kinase), a member of the tobacco MAPK (mitogen-activated protein kinase) family that is activated by various other stress stimuli. The activation of the 40-kD protein kinase is rapid and dose-dependent. Other osmolytes such as Pro and sorbitol activate these two kinases with similar kinetics. The activation of 40-kD protein kinase is specific for hyperosmotic stress, as hypotonic stress does not activate it. Therefore, this 40-kD kinase was named HOSAK (high osmotic stress-activated kinase). HOSAK is a Ca(2+)-independent kinase and uses myelin basic protein (MBP) and histone equally well as substrates. The kinase inhibitor K252a rapidly activates HOSAK in tobacco cells, implicating a dephosphorylation mechanism for HOSAK activation. Activation of both SIPK and HOSAK by high osmotic stress is Ca(2+) and abscisic acid (ABA) independent. Furthermore, mutation in SOS3 locus does not affect the activation of either kinase in Arabidopsis seedlings. These results suggest that SIPK and 40-kD HOSAK are two new components in a Ca(2+)- and ABA-independent pathway that may lead to plant adaptation to hyperosmotic stress.     

A 42-kilodalton annexin-like protein is associated with plant vacuoles.

A 42-kD, calcium-dependent, membrane-binding protein (VCaB42) was associated with partially purified vacuole membrane. Membrane-dissociation assays indicated that VCaB42 binding to vacuole membranes was selective for calcium over other cations and that 50% of VCaB42 remained membrane bound at 61 +/- 11 nM free calcium. A 13-amino acid sequence obtained from VCaB42 showed 85% similarity with the endonexin fold, a sequence found in the annexin family of proteins that is thought to be essential for calcium and lipid binding. The greatest similarity in amino acid sequence was observed with annexin VIII (VAC-beta). The calcium-binding properties and sequence similarities suggest that VCaB42 is a member of the annexin family of calcium-dependent, membrane-binding proteins. Functional assays for VCaB42 on vacuole membrane transport processes indicated that it did not significantly affect the initial rate of calcium uptake into vacuole membrane vesicles. Because VCaB42 is vacuole localized (likely on the cytosolic surface of the vacuole) and is 50% dissociated within the physiological range of cytosolic free calcium, we hypothesize that this protein is a sensor that monitors cytosolic calcium levels and transmits that information to the vacuole.     

Cks1 is required for G(1) cyclin-cyclin-dependent kinase activity in budding yeast

p13(suc1) (Cks) proteins have been implicated in the regulation of cyclin-dependent kinase (CDK) activity. However, the mechanism by which Cks influences the function of cyclin-CDK complexes has remained elusive. We show here that Cks1 is required for the protein kinase activity of budding yeast G(1) cyclin-CDK complexes. Cln2 and Cdc28 subunits coexpressed in baculovirus-infected insect cells fail to exhibit protein kinase activity towards multiple substrates in the absence of Cks1. Cks1 can both stabilize Cln2-Cdc28 complexes and activate intact complexes in vitro, suggesting that it plays multiple roles in the biogenesis of active G(1) cyclin-CDK complexes. In contrast, Cdc28 forms stable, active complexes with the B-type cyclins Clb4 and Clb5 regardless of whether Cks1 is present. The levels of Cln2-Cdc28 and Cln3-Cdc28 protein kinase activity are severely reduced in cks1-38 cell extracts. Moreover, phosphorylation of G(1) cyclins, which depends on Cdc28 activity, is reduced in cks1-38 cells. The role of Cks1 in promoting G(1) cyclin-CDK protein kinase activity both in vitro and in vivo provides a simple molecular rationale for the essential role of CKS1 in progression through G(1) phase in budding yeast.     

Identification of a human gene (HCK) that encodes a protein-tyrosine kinase and is expressed in hemopoietic cells.

We have isolated cDNAs representing a previously unrecognized human gene that apparently encodes a protein-tyrosine kinase. We have designated the gene as HCK (hemopoietic cell kinase) because its expression is prominent in the lymphoid and myeloid lineages of hemopoiesis. Expression in granulocytic and monocytic leukemia cells increases after the cells have been induced to differentiate. The 57-kilodalton protein encoded by HCK resembles the product of the proto-oncogene c-src and is therefore likely to be a peripheral membrane protein. HCK is located on human chromosome 20 at bands q11-12, a region that is affected by interstitial deletions in some acute myeloid leukemias and myeloproliferative disorders. Our findings add to the diversity of protein-tyrosine kinases that may serve specialized functions in hemopoietic cells, and they raise the possibility that damage to HCK may contribute to the pathogenesis of some human leukemias.     

Analysis of a tyrosine-specific protein kinase activity associated with the retroviral erbB oncogene product

The transforming protein erbB of avian erythroblastosis virus (AEV) has considerable sequence homology with the epidermal growth factor (EGF) and appears to represent a truncated form of this receptor. The sequence of the erbB gene is furthermore related to that of other viral transforming genes such as src, fps, yes or abl. The transforming proteins of these src-related oncogenes as well as receptors for EGF, platelet-derived growth factor (PDGF), and insulin are associated with tyrosine-specific protein kinases. It has been difficult to demonstrate this activity for the erbB protein. To analyze the erbB gene product, we prepared polyclonal antibodies against a bacterially expressed erbB DNA restriction fragment (BamHI/BamHI). The antiserum is shown to immunoprecipitate the erbB protein from AEV-transformed chicken fibroblasts and also recognizes the EGF receptor protein. Both proteins become phosphorylated in vitro on tyrosine residues upon the addition of [gamma-32P]ATP. The protein kinase activity is low compared to other oncogene-specific kinases. This is not due to kinase blocking by the serum, because erbB carboxyterminal synthetic peptide antibodies give rise to low levels of protein kinase activity as well indicating that this may be a characteristic property of erbB in vitro.     

Adenovirus E1A is associated with a serine/threonine protein kinase.

The adenovirus E1A proteins form stable protein complexes with a number of cellular proteins, including cyclin A and the product of the retinoblastoma susceptibility gene. We have been interested in learning about the function of proteins associated with E1A and therefore looked for an enzymatic activity present in E1A complexes. We found a serine/threonine kinase activity that phosphorylates two proteins bound to E1A, the 107- and 130-kDa (107K and 130K) proteins. The kinase also phosphorylates histone H1 added as an exogenous substrate. The kinase activity is cell cycle regulated, being most active in S and G2/M-phase cells. The timing of phosphorylation of the 107K protein in vitro correlates with the phosphorylation pattern of the 107K protein in vivo. A variety of genetic and immunochemical approaches indicate that the activity is probably not due to the E1A-associated 300K, 130K, 107K, or pRB protein. Although we have not established the identity of the kinase, we present evidence that the kinase activity is consistent with phosphorylation by p34cdc2 or a related kinase.