A thousand and one protein kinases.
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Animals↗
Biomedical subjects
T Hunter
Publications and source records attributed to T Hunter.
cDNA sequence and tissue distribution of the mRNA for bovine and murine p11, the S100-related light chain of the protein-tyrosine kinase substrate p36 (calpactin I).
We have isolated and sequenced cDNA clones of bovine and murine p11 mRNAs. The nonpolyadenylated mRNAs are predicted to be 614 and 600 nucleotides, respectively. The p11 mRNAs both contain a 291 nucleotide open reading frame, preceded by a 5'-untranslated region of 73 nucleotides in bovine p11 mRNA and of 68 nucleotides in murine p11 mRNA. The deduced bovine p11 amino acid sequence is identical to the previously published partial bovine and complete porcine p11 protein sequence except for an additional COOH-terminal lysine residue. The bovine and murine p11 proteins are 92% homologous, whereas at the nucleotide level the conservation is 89% in the coding region and 75% in the 3'-untranslated region. Southern analysis of murine genomic DNA detected a single p11 gene, less than 10 kilobase pairs in size, containing as many as three introns. The p11 gene has been assigned to mouse chromosome 3 by analysis of interspecific hybrid cell panels and recombinant inbred mouse strains. The p11 gene is closely linked to the Xmmv-65 endogenous leukemia virus env gene and the guanylate binding protein-1 gene. Northern analyses of RNAs from mouse tissues and cell lines indicated that p11 mRNA levels vary widely. They are very low in liver, heart, and testes, moderate in brain, spleen, and thymus, and high in kidney, intestine, and lung. Analysis of the same RNA samples for p36 mRNA levels showed that expression of p11 and p36 mRNAs is not always coordinated. Brain and the mouse embryonal carcinoma cell line F9 contain moderate to high levels of p11 mRNA with very low levels of p36 mRNA. Sequence homology between p11 and the S100 proteins, and the serum-induced 2A9 gene product, as well as possible functions of p11 are discussed.
Amino Acid Sequence↗
A tail of two src's: mutatis mutandis.
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Animals↗
Isolation and characterization of two distinct forms of protein kinase C.
Protein kinase C (Ca2+- and phospholipid-dependent protein kinase) has been purified from rat brain by a three-step, 18-h procedure resulting in the isolation of milligram quantities of enzyme. Unlike previous preparations from published protocols, which yield a single polypeptide, this procedure yields a protein which consists of a 78/80-kDa doublet upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The two polypeptides have been characterized with respect to structure and function and are very similar in both regards. However, the two forms can be distinguished immunologically by polyclonal antisera generated against purified protein kinase C. The 78- and 80-kDa proteins do not appear to be related to one another by proteolytic cleavage or by differential phosphorylation, although the two purified proteins do contain stoichiometric amounts of phosphate. The 78- and 80-kDa polypeptides therefore appear to represent two distinct forms of protein kinase C, thus providing evidence for the existence of multiple isozymes of this key regulatory protein.
Animals↗
Hemophilus aphrophilus vertebral osteomyelitis: a case report and literature review.
We report a 60-yr-old diabetic female who developed vertebral osteomyelitis and discitis due to Hemophilus aphrophilus, and review three previously reported cases of this uncommon infection. Predisposing factors, clinical features, and treatment of H. aphrophilus vertebral osteomyelitis are discussed.
Female↗
Decreased bioactivity of circulating prolactin in patients with rheumatoid arthritis.
Growth hormone and prolactin (PRL) were measured by radio-immunoassay in sera of 36 patients with rheumatoid arthritis (24 females and 12 males) and of 50 normal blood donors (25 males and 25 females). The bioactivity of PRL was also determined for both groups, using the Nb2 rat lymphoma proliferation assay. Although no significant difference was found between serum PRL values of patients and controls when determined by radio-immunoassay, the bioactivity of PRL was significantly decreased in patients' sera, when compared to values obtained in age and sex-matched controls. This decreased PRL bioactivity could not be attributed to drug treatments administered to the patients examined.
Arthritis, Rheumatoid↗
Immunological evidence for two physiological forms of protein kinase C.
Our recently described purification scheme for rat brain protein kinase C yields an enzyme consisting of a 78/80-kilodalton (kDa) doublet upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (submitted for publication). Antisera against this preparation were raised in two rabbits. One of the antisera detected only the 80-kDa component by immunoblotting of purified protein kinase C and immunoprecipitated an 80-kDa [35S]methionine-labeled protein from a variety of human, rodent, and bovine cells, which was shown to represent protein kinase C by comparative one-dimensional peptide mapping. In contrast, the second antiserum detected both 78- and 80-kDa enzyme forms by immunoblotting and immunoprecipitated a [35S]methionine-labeled 78/80-kDa doublet from mammalian cells. One-dimensional peptide maps of these 78- and 80-kDa proteins were similar to those derived from the 78- and 80-kDa forms of purified protein kinase C, respectively. The two forms were not related by either partial proteolysis or differential phosphorylation, showing that two distinct forms of this enzyme exist in mammalian cells. Treatment of mouse B82 L cells with 2.5 micrograms of 12-O-tetradecanoylphorbol-13-acetate (TPA) per ml for 18 h resulted in complete loss of immunoprecipitable protein kinase C with a half time of disappearance of 48 min. Since the normal half-life of protein kinase C was greater than 24 h and the biosynthetic rate of the protein was not decreased after 18 h by TPA treatment, TPA induces down-regulation by increasing the degradation rate of the enzyme. Treatment of cells with 50 ng of TPA per ml followed by resolution of the membrane and cytosol in the presence of ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA) promoted an apparent translocation of both 78- and 80-kDa proteins from the cytosol to the membrane fraction. A similar translocation was effected by cell lysis in the presence of Ca2+, indicating the subcellular localization of protein kinase C to be sensitive to the presence of both activators and micromolar amounts of Ca2+.
Amino Acids↗
Alterations in pp60c-src accompany differentiation of neurons from rat embryo striatum.
Cultured neurons from rat embryo striatum were found to contain two structurally distinct forms of pp60c-src. The 60-kilodalton (kDa) form appeared similar to pp60c-src from cultured rat fibroblasts or astrocytes. The 61-kDa form was specific to neurons and differed in the NH2-terminal 18 kDa of the molecule. In undifferentiated neurons the predominant phosphorylated species of pp60c-src was the fibroblast form. Upon differentiation, a second phosphorylated form of pp60c-src was detected. This form had two or more additional sites of serine phosphorylation within the NH2-terminal 18-kDa region of the molecule, one of which was Ser-12. The specific protein-tyrosine kinase activity of the total pp60c-src population increased 14-fold, as measured by autophosphorylation, or 7-fold, as measured by phosphorylation of an exogenous substrate, as striatal neurons differentiated. This elevation in protein kinase activity occurred without a detectable decrease in Tyr-527 phosphorylation or increase in Tyr-416 phosphorylation. Our results support the idea that the expression of the neuron-specific form of pp60c-src and the increase in specific protein kinase activity may be important for neuronal differentiation.
Animals↗
Phorbol ester-induced down-regulation of protein kinase C abolishes vasopressin-mediated responses in rat anterior pituitary cells.
The role of protein kinase C (PKC) in the multihormonally regulated ACTH secretory responses of rat anterior pituitary cells was examined in control cells or after pretreatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of PKC. Using affinity-purified polyclonal antiserum raised against purified rat brain PKC, immunoprecipitable PKC was demonstrated in [35S]methionine-labeled cells appearing as a doublet of 78/80 kilodaltons. Long-term treatment (24 h) of cells with 0.6 microM TPA caused the specific loss of immunologically reactive PKC. Consistently, TPA pretreatment decreased the amount of phosphatidylserine-dependent protein kinase activity measured in vitro by 90%. In control cells, vasopressin (AVP) stimulated ACTH secretion and potentiated ACTH secretion stimulated by CRF. After a 24-h treatment with 0.6 microM TPA, secretory responses to AVP and the potentiating effect of AVP on CRF action were completely abolished. In contrast, CRF action on ACTH secretion, thought to be mediated by cAMP, was unaffected. Similarly, forskolin- and 8 bromo-cAMP-induced ACTH secretion remained unchanged after TPA pretreatment. These results indicate a crucial role for PKC in mediating the effects of AVP on ACTH secretion and on the potentiating action of AVP on CRF-induced secretion from corticotropic cells of the anterior pituitary.
Animals↗
Regulation of protein kinase C by activators, Ca2+, and phosphorylation.
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Animals↗
Substrate specificity of protein kinase C. Use of synthetic peptides corresponding to physiological sites as probes for substrate recognition requirements.
Although the Ca2+/phospholipid-dependent protein kinase, protein kinase C, has a broad substrate specificity in vitro, the enzyme appears considerably less promiscuous in vivo. To date only a handful of proteins have been identified as physiological substrates for this protein kinase. In order to determine the basis for this selectivity for substrates in intact cells, we have probed the substrate primary sequence requirements of protein kinase C using synthetic peptides corresponding to sites of phosphorylation from four of the known physiological substrates. We have also identified the acetylated N-terminal serine of chick muscle lactate dehydrogenase as an in vitro site of phosphorylation for this protein kinase. These comparative studies have demonstrated that, in vivo, the enzyme exhibits a preference for one basic residue C-terminal to the phosphorylatable residue, as in the sequence: Ser/Thr-Xaa-Lys/Arg, where Xaa is usually an uncharged residue. Additional basic residues, both N and C-terminal to the target amino acid, enhance the Vmax and Km parameters of phosphorylation. None of the peptides based on physiological phosphorylation sites of protein kinase C was an efficient substrate of cAMP-dependent protein kinase, emphasizing the distinct site-recognition selectivities of these two pleiotropic protein kinases. The favorable kinetic parameters of several of the synthetic peptides, coupled with their selectivity for phosphorylation by protein kinase C, will facilitate the assay of this enzyme in the presence of other protein kinases in tissue and cell extracts.
Binding Sites↗
Association of the S-100-related calpactin I light chain with the NH2-terminal tail of the 36-kDa heavy chain.
Calpactin I, a Ca2+- and phospholipid-binding cytoskeletal protein, which serves as a major substrate of protein-tyrosine kinases, was isolated from bovine intestine and lung as a species containing two 36-kDa heavy chains and two 10-kDa light chains. The heavy chain is comprised of two distinct domains which can be identified by limited proteolysis: a COOH-terminal 33-kDa core, which contains the Ca2+- and phospholipid-binding sites, and an NH2-terminal tail, which contains the major site of phosphorylation by pp60v-src. To determine the site of association of the light chain on the heavy chain, we analyzed the association states of the light chain, core, and tail by sucrose gradient centrifugation after limited chymotryptic digestion. The core was not detected in higher Mr complexes with the light chain, and the tail cosedimented with a light chain dimer. The tail, isolated from chymotryptic digests and radiolabeled with 125I, was found to form a specific complex with the light chain, but not the core. The authentic tail and a synthetic peptide corresponding to residues 1-29 of the calpactin I heavy chain were both able to specifically inhibit the reassociation between heavy and light chain, whereas a synthetic peptide corresponding to residues 15-33 was inactive. These results suggest that the tail may serve as a site of regulation by light chain or phosphorylation.
Animals↗
Primary structure of bovine calpactin I heavy chain (p36), a major cellular substrate for retroviral protein-tyrosine kinases: homology with the human phospholipase A2 inhibitor lipocortin.
An amplified Okayama-Berg plasmid cDNA library was constructed from total poly(A)+ RNA isolated from the Madin-Darby bovine kidney cell line MDBK. This library was screened with a partial murine calpactin I heavy chain (p36) cDNA clone, the identification of which was based on bovine p36 tryptic peptide sequences generated during the course of these studies. The largest p36 cDNA insert (p36/6 of 1.6 kilobase pairs) was fully sequenced by the dideoxy method. The DNA sequence of this insert had an open reading frame of 1014 base pairs and coded for a protein with a molecular weight of 38 481. The deduced protein sequence of 338 residues was concordant with 173 residue positions of p36 determined at the protein level. The 5'- and 3'-ends of p36/6 contained 54 and 307 base pairs of untranslated sequence, respectively. Examination of poly(A)+ RNA prepared from the Madin-Darby cell line indicated a p36 mRNA species of about 1.6 kilobases. Four regions of internal homology, each about 70 amino acid residues in length, were observed in the deduced protein sequence for p36. Thirty-three of the 70 residue positions were conserved in at least three of the four repeating units. A comparison of derived amino acid sequence for bovine p36 with that previously determined for human lipocortin [Wallner, B. P., Mattaliano, R. J., Hession, C., Cate, R. L., Tizard, R., Sinclair, L. K., Foeller, C., Chow, E. P., Browning, J. L., Ramachandran, K. L., & Pepinsky, R. B. (1986) Nature (London) 320, 77-81] revealed extensive homology (66% overall) and the presence of four repetitive regions in the lipocortin structure.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
The cDNA sequence for the protein-tyrosine kinase substrate p36 (calpactin I heavy chain) reveals a multidomain protein with internal repeats.
We have isolated and sequenced a full-length cDNA clone for the protein-tyrosine kinase substrate p36 (calpactin I heavy chain). This sequence predicts a 339 amino acid (Mr 38,493) protein containing an N-terminal region of 20 amino acids, known to interact with a 10 kd protein (light chain), and a C-terminal region, found to contain two Ca2+/phospholipid-binding sites, that can be aligned as four 70 amino acid repeats. A single p36 gene was detected in the mouse genome, and a major p36 mRNA of 1.6 kb was found to be expressed in different mouse tissues. Unexpectedly, p36 and the phospholipase A2 inhibitor lipocortin I were found to be 50% identical in sequence over the C-terminal 300 residues. The function of p36 and its relation to other proteins are discussed.
Amino Acid Sequence↗
Tyr527 is phosphorylated in pp60c-src: implications for regulation.
The Rous sarcoma virus oncogene product, pp60v-src, transforms cultured fibroblasts but its corresponding proto-oncogene product, pp60c-src, does not. Both proteins are known to be protein-tyrosine kinases. Published results suggest that the kinase activity of pp60c-src is inhibited relative to that of pp60v-src, due perhaps to phosphorylation of a tyrosine in pp60c-src that is not phosphorylated in pp60v-src. In this study, it was observed that the tyrosine phosphorylated in pp60c-src is Tyr527, six residues from the COOH-terminus of the protein. The region of pp60c-src from residue 515 to the COOH-terminus, including Tyr527, has been replaced with a different sequence in pp60v-src. Thus, the increase in transforming ability and kinase activity that occurred in the genesis of pp60v-src may have resulted from the loss of a tyrosine involved in negative regulation.
Animals↗
Early phosphorylation events following the treatment of Swiss 3T3 cells with bombesin and the mammalian bombesin-related peptide, gastrin-releasing peptide.
Bombesin and the related mammalian peptides, such as gastrin-releasing peptide (GRP), are potent mitogens for some fibroblast cell lines. Here we have examined the bombesin- and GRP-mediated changes in the phosphorylation of proteins in Swiss 3T3 cells and compared these to the events observed after platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and tumor promoter treatment. In agreement with previous reports, bombesin, GRP and PDGF, but not EGF, increased the activity of protein kinase C. This was assayed by an inhibition of [125I]EGF binding, stimulation in phosphorylation of pp60c-src on serine 12 and stimulation in phosphorylation of a group of 80 kd proteins. The different phosphorylated forms of the 80 kd proteins were examined by tryptic peptide mapping and shown to contain multiple phosphorylation sites. An investigation of the tyrosine phosphorylation events following mitogen treatment revealed a significant difference between PDGF and the bombesin peptides. PDGF treatment caused a marked increase in total cellular phosphotyrosine levels, and tyrosine phosphorylation both of known substrates and its own receptor. In contrast, bombesin and GRP treatments resulted in only a weak or undetectable increase in tyrosine phosphorylation of total cellular protein or known substrates. In this respect bombesin and GRP were more similar to EGF. The fact that the bombesin peptides do not induce a phosphorylation response identical with either PDGF or EGF suggests that there is not a single common signal pathway which is activated by all these mitogens.
Animals↗
Identification of phosphoproteins correlated with proliferation and cell cycle arrest in Saccharomyces cerevisiae: positive and negative regulation by cAMP-dependent protein kinase.
Recent genetic and biochemical studies of two mutants of the cAMP pathway in yeast, cyr1 and bcy1, have demonstrated that cAMP-dependent protein phosphorylation plays a major regulatory role in the control of proliferation and differentiation. As a first step in examining this regulatory system in more detail and in identifying the protein substrates of cAMP-dependent protein kinase, we have analyzed phosphoprotein patterns in the mutants cyr1-2(ts) and bcy1 by two-dimensional polyacrylamide gel electrophoresis. Our analysis has revealed several proteins whose phosphorylation is controlled positively or negatively by the cAMP pathway in yeast. The presence of some of these phosphoproteins was directly associated with proliferation (positive regulation), while that of others was correlated with cell cycle arrest (negative regulation). The phosphoprotein patterns of cyr1-2(ts) temperature-arrested cells, and nitrogen (NH+4)-starved cells, were strikingly similar, suggesting that response to NH+4 is mediated in part by adenylate cyclase. Phosphoproteins whose presence correlated with cell cycle arrest were found to be phosphorylated on serine and threonine residues, while the major phosphoproteins present predominantly in proliferating cells were phosphorylated only on serine residues. None of the greater than 20 phosphoproteins we examined contained phosphotyrosine under either growth condition.
Cell Cycle↗
The protein-tyrosine kinase substrate, p81, is homologous to a chicken microvillar core protein.
p81, a protein-tyrosine kinase substrate previously identified in epidermal growth factor-treated A431 cells, is demonstrated to be homologous to ezrin, an 80-kD component of microvillar core proteins. p81 has been characterized using antiserum raised against purified chicken intestinal ezrin. p81, located by indirect immunofluorescent staining, is concentrated in surface projections of A431 cells such as microvilli and retraction fibers. None of the conditions of biochemical cell fractionation tested completely solubilizes p81; the insoluble p81 partitions as if associated with the cytoskeleton. The soluble form of p81 behaves as a monomer in all extraction procedures studied. EGF-stimulated phosphorylation of p81 does not appear to change its intracellular location. p81 exhibits a wide tissue distribution with highest levels of expression in small intestine, kidney, thymus, and lung. Intermediate levels are found in spleen, thymus, lymph nodes, and bone marrow, with low levels in brain, heart, and testes. p81 is undetectable in muscle and liver. In A431 cells, p81 is phosphorylated on serine and threonine residues. Upon EGF treatment, approximately 10% of p81 becomes phosphorylated on tyrosine, and the phosphorylation of threonine residues increases.
Animals↗