Tyrosyl Protein Kinase Research Articles

Enhancement of HIV-1-Induced Syncytium Formation in T Cells by the Tyrosyl Kinase p56lck

The CD4 glycoprotein is the primary cellular receptor for human immunodeficiency virus type 1 (HIV-1) and has also been reported to be physically associated with p56lck, a tyrosyl protein kinase. p56lckis a member of thesrcfamily of nonreceptor protein-tyrosine kinases and is expressed predominantly in T lymphocytes. Our objective was to study the effect of p56lckon the biology of HIV-1. For this purpose, we have stably transfected two human p56lck-negative T cell lines (C8166-45 and MT-2) with plasmids encoding for this cellular protein. Following coculture with HIV-1-infected cells or infection with cell-free virus, p56lck-expressing cell lines showed a greater propensity for virus-mediated syncytium formation than parental p56lck-negative cells. The enhancement of HIV-1-induced syncytium formation was not associated with the kinase activity of p56lck, as demonstrated by experiments using a kinase-deficient mutant. However, the physical interaction between CD4 and p56lckwas shown to be necessary to obtain the enhancement of syncytium formation since a mutated version of p56lck, which is deficient in its capacity to associate with CD4, did not lead to an increase in virus-mediated cell-to-cell fusion events. Finally, we determined that cells transfected with wild-type and kinase-negative mutant p56lckshowed a reduced rate of CD4 endocytosis compared to parental p56lck-negative cells. Together, these results suggest that p56lckcan be seen as an accessory molecule facilitating HIV-1-mediated syncytium formation in T cells by a mechanism involving the stabilization of the CD4 molecule at the cell surface.

The SH3 domain of Src tyrosyl protein kinase interacts with the N-terminal splice region of the PDE4A cAMP-specific phosphodiesterase RPDE-6 (RNPDE4A5).

The PDE4A (type IV) cAMP-specific, rolipram-inhibited phosphodiesterase RPDE-6 (RNPDE4A5), when transiently expressed in COS7 cells, could be complexed with the v-Src-SH3 domain expressed as a glutathione S-transferase (GST) fusion protein. RPDE-6 did not interact with GST itself. This complex was not disrupted by treatment with high NaCl concentration together with Triton X-100. Interaction was apparently determined by the N-terminal splice region of RPDE-6, as the PDE4A splice variant RPDE-39, which differs from RPDE-6 at the extreme N-terminus, failed to associate with v-Src-SH3; met26RD1 (where RD1 is rat 'dunc-like' PDE), which has the N-terminal splice region deleted, failed to associate with v-Src-SH3, and the association of RPDE-6 and v-Src-SH3 was blocked by a fusion protein formed from the N-terminal splice region. RDPE-6 showed binding to GST fusion proteins of both the intact Src kinase and an SH2-SH3 construct but did not bind to the Src-SH2 domain or to the adaptor protein Grb-2. RPDE-6 could be co-immunoprecipitated from cytosol extracts of transfected cells by using anti-Src antiserum. RPDE-6 exhibited selectivity in binding to the SH3 domains of c-Abl, Crk, Csk, Lck, Lyn, Fyn and v-Src, with binding to the SH3 regions of the Src-related tyrosyl kinases Lyn and Fyn being the most effective. The binding of RPDE-6 to the SH3 domains of Crk, Csk and Lck led to a marked reduction in PDE activity, but no change was apparent in complexes with other species. Endogenous RPDE-6 from brain, but not endogenous RPDE-39 from testis, bound to the Src-SH3 domain. We suggest that the PDE4A splice variant RPDE-6 has a propensity for interaction with selective SH3 domains, in particular those from Src and the Src-related tyrosyl kinases Lyn and Fyn. This interaction seems to be governed by alternative splicing of the PDE4A gene, because RPDE-39, a splice variant that lacks the proline-rich N-terminal splice region of RPDE-6, does not interact with these SH3 domains. It is proposed that the binding site on RPDE-6 for SH3 domains lies within the unique first 102 residues of its N-terminal splice domain, where two motifs representing Class I SH3 binding sites with selectivity for Src kinase SH3 domains can be identified and one motif for a putative Class II SH3 binding site.

Inactivation of a redox-sensitive protein phosphatase during the early events of tumor necrosis factor/interleukin-1 signal transduction.

Isoforms of heat shock protein (Hsp) 27 were used as intracellular markers to study tumor necrosis factor/interleukin-1 (TNF/IL-1) regulation of protein phosphatases in primary human fibroblasts. These isoforms were rapidly phosphorylated to varying degrees when fibroblasts were treated with either TNF, IL-1, okadaic acid, calyculin A, ARS, epidermal growth factor, fibroblast growth factor, H2O2, buthionine sulfoximine, N-ethylmaleimide, diethylmaleimide, or iodoacetate. However, inhibitors of protein kinases A and C, tyrosyl protein kinases, and general protein kinases had no effect on the enhanced phosphorylation of these isoforms in TNF, IL-1, okadaic acid, or calyculin A-stimulated cells, suggesting that the activation of protein kinases by itself is insufficient to produce these changes. Isoforms of 32P-labeled Hsp27 were dephosphorylated during cold-chases with excess phosphate in the absence but not in the presence of TNF/IL-1 or inhibitors of protein phosphatases suggesting that inactivation of protein phosphatase(s) plays a role in TNF/IL-1 signal transduction. Assays of phosphatase activity of cytosolic fractions from TNF or okadaic acid treated human fibroblasts showed an inactivation of protein phosphatase activity against the 32P-labeled Hsp27 protein substrates. In vitro assays of partially purified phosphatase activity from primary human fibroblasts with Hsp27 substrate also showed the protein phosphatase activity to be inhibited by ARS. Like okadaic acid, ARS mimics TNF in inducing specific patterns of cellular protein phosphorylation. Taken together these findings are consistent with the hypothesis that a SH-dependent protein phosphatase is inactivated during the early events of TNF/IL-1 signal transduction, hence inhibitors of protein phosphatases and SH modifying compounds can mimic the early effects of TNF/IL-1 on cells.

Tyrosyl phosphorylation and activation of MAP kinases by p56lck.

T cell signaling via the CD4 surface antigen is mediated by the associated tyrosyl protein kinase p56lck. The 42-kilodalton mitogen-activated protein (MAP) kinase (p42mapk) was tyrosyl-phosphorylated and activated after treatment of the murine T lymphoma cell line 171CD4+, which expresses CD4, with antibody to CD3. Treatment of the CD4-deficient cell line 171 with the same antibody did not result in phosphorylation or activation of p42mapk. Purified p56lck both tyrosyl-phosphorylated and stimulated the seryl-threonyl phosphotransferase activity of purified p44mpk, a MAP kinase isoform from sea star oocytes. A synthetic peptide modeled after the putative regulatory phosphorylation site in murine p42mapk (Tyr185) was phosphorylated by p56lck with a similar Vmax, but a fivefold lower Michaelis constant (Km) than a peptide containing the Tyr394 autophosphorylation site from p56lck. MAP kinases may participate in protein kinase cascades that link Src family protein-tyrosyl kinases to seryl-threonyl kinases such as those encoded by rsk and raf, which are putative substrates of MAP kinases.

Phosphorylation and Activation of Epidermal Growth Factor Receptors in Cells Transformed by the src Oncogene

Because functionally significant substrates for the tyrosyl protein kinase activity of pp60v-src are likely to include membrane-associated proteins involved in normal growth control, we have tested the hypothesis that pp60v-src could phosphorylate and alter the signaling activity of transmembrane growth factor receptors. We have found that the epidermal growth factor (EGF) receptor becomes constitutively phosphorylated on tyrosine in cells transformed by the src oncogene and in addition displays elevated levels of phosphoserine and phosphothreonine. High-performance liquid chromatography phosphopeptide mapping revealed two predominant sites of tyrosine phosphorylation, both of which differed from the major sites of receptor autophosphorylation; thus, the src-induced phosphorylation is unlikely to occur via an autocrine mechanism. To determine whether pp60v-src altered the signaling activity of the EGF receptor, we analyzed the tyrosine phosphorylation of phospholipase C-gamma, since phosphorylation of this enzyme occurs in response to activation of the EGF receptor but not in response to pp60v-src alone. We found that in cells coexpressing pp60v-src and the EGF receptor, phospholipase C-gamma was constitutively phosphorylated, a result we interpret as indicating that the signaling activity of the EGF receptor was altered in the src-transformed cells. These findings suggest that pp60v-src-induced alterations in phosphorylation and function of growth regulatory receptors could play an important role in generating the phenotypic changes associated with malignant transformation.

Phosphorylation and activation of epidermal growth factor receptors in cells transformed by the src oncogene.

Because functionally significant substrates for the tyrosyl protein kinase activity of pp60v-src are likely to include membrane-associated proteins involved in normal growth control, we have tested the hypothesis that pp60v-src could phosphorylate and alter the signaling activity of transmembrane growth factor receptors. We have found that the epidermal growth factor (EGF) receptor becomes constitutively phosphorylated on tyrosine in cells transformed by the src oncogene and in addition displays elevated levels of phosphoserine and phosphothreonine. High-performance liquid chromatography phosphopeptide mapping revealed two predominant sites of tyrosine phosphorylation, both of which differed from the major sites of receptor autophosphorylation; thus, the src-induced phosphorylation is unlikely to occur via an autocrine mechanism. To determine whether pp60v-src altered the signaling activity of the EGF receptor, we analyzed the tyrosine phosphorylation of phospholipase C-gamma, since phosphorylation of this enzyme occurs in response to activation of the EGF receptor but not in response to pp60v-src alone. We found that in cells coexpressing pp60v-src and the EGF receptor, phospholipase C-gamma was constitutively phosphorylated, a result we interpret as indicating that the signaling activity of the EGF receptor was altered in the src-transformed cells. These findings suggest that pp60v-src-induced alterations in phosphorylation and function of growth regulatory receptors could play an important role in generating the phenotypic changes associated with malignant transformation.

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

Because examination of regulatory trans-phosphorylations can help elucidate the cellular functions of tyrosyi protein kinases, we have investigated the effects of phosphorylation by casein kinase-1 on the activity of the p40 tyrosyi 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 a Phosphotyrosyl-protein Phosphatase in Mouse Epidermis

Binding of epidermal growth factor (EGF) stimulates tyrosyl protein kinase activity of its receptor in the epidermis. This tyrosine residue phosphorylation is thought to be one mechanism by which EGF mediates its effects such as growth stimulation. To modulate a cellular response to EGF, an enzyme which dephosphorylates phosphotyrosyl residues should be present to oppose the effect of the tyrosyl kinase activity of the EGF receptor. We have identified an enzyme in the neonatal mouse epidermis which has the ability to dephosphorylate tyrosyl residues in vitro on EGF receptors. This phosphatase is a soluble protein with a molecular weight greater than 10,000 daltons and shows optimum activity at neutral pH. This epidermal tyrosyl protein phosphatase is not inhibited by tartrate, ATP, and micromolar levels of zinc, but is inhibited by millimolar levels of zinc, magnesium, manganese, and fluoride. Unlike other well-known phosphotyrosyl phosphatases, alkaline phosphatase, and calcineurin, this enzyme is not inhibited by EDTA. Thus, we have identified and partially characterized a possibly unique phosphotyrosyl phosphatase from the epidermis.

Partial purification of a tyrosyl protein kinase from rat liver cytosol by affinity chromatography on poly(Glu:Tyr)-linked Sepharose 4B

Partial purification of a tyrosyl protein kinase from rat liver cytosol by affinity chromatography on poly(Glu:Tyr)-linked Sepharose 4B

The stereospecificity of protein kinases

To test whether cellular protein kinases exist that phosphorylate d-amino acid residues, a method was developed for separating O-phospho- d-serine from O-phospho- l-serine and O-phospho- l-tyrosine from O-phospho- d-tyrosine. This was accomplished by converting these amino acids to the l-leucyl dipeptide derivatives followed by separation of the diastereomers by anion-exchange high-performance liquid chromatography. The enantiomeric content of these d- and l-residues were measured in hydrolysates of 32P-labeled proteins produced by the protein kinases of human erythrocytes and the tyrosyl protein kinase of the Abelson leukemia virus. We found no measurable d-phosphoserine in erythrocyte membrane proteins under conditions where a 1% content of this residue relative to l-phosphoserine would have been detected. These values can be used to place an upper hypothetical limit on the fraction of erythrocyte protein kinase activity that is specific for serine residues in the d-configuration. In separate experiments, we examined the specificity of the tyrosyl protein kinases. We found that all of the phosphotyrosine that we isolated from the erythrocyte band 3 NH 2-terminal fragment and from the autophosphorylation of the Abelson virus tyrosyl kinase was in the l-configuration.

(Glu62, Ala30, Tyr8)n serves as high-affinity substrate for tyrosylprotein sulfotransferase: a Golgi enzyme.

Tyrosylprotein sulfotransferase, the enzyme catalyzing the sulfation of proteins on tyrosine residues, was characterized by using the acidic polymer containing tyrosine (Glu62, Ala30, Tyr8)n (referred to as Glu,Ala,Tyr) as exogenous "protein" substrate. After subcellular fractionation of a bovine adrenal medulla homogenate, tyrosylprotein sulfotransferase activity was found to be highest in fractions enriched in Golgi membrane vesicles. Tyrosylprotein sulfotransferase required the presence of a nonionic detergent for sulfation of exogenous Glu,Ala,Tyr, indicating an orientation of the catalytic site of the enzyme toward the Golgi lumen. Tyrosylprotein sulfotransferase was solubilized by Triton X-100, suggesting that the enzyme was tightly associated with the Golgi membrane, possibly as an integral membrane protein. The apparent Golgi localization of tyrosylprotein sulfotransferase was supported by the observation that tyrosine sulfation of proteins in intact cells was blocked by monensin and was in line with previous observations that all tyrosine-sulfated proteins known so far are secretory. Glu,Ala,Tyr was found to have a very high affinity for tyrosylprotein sulfotransferase (apparent Km, 300 nM), similar to that reported for certain tyrosylprotein kinases. While this may suggest some similarity between these enzymes, the Golgi localization of tyrosylprotein sulfotransferase segregates tyrosine sulfation from the sites of tyrosine phosphorylation of proteins in the intact cell. If, however, tyrosylprotein sulfotransferase was allowed to react with cytoplasmic proteins by using a nonionic detergent, tyrosine sulfation of tubulin was observed.

Inhibition of the activation and catalytic activity of insulin receptor kinase by zinc and other divalent metal ions.

The autophosphorylation reaction responsible for conversion of insulin receptor (from human placenta) to an active tyrosyl-protein kinase was shown to be inhibited by Zn2+ and other divalent metal ions. The order of inhibitory potency was found to be Cu2+ greater than Zn2+, Cd2+ greater than Co2+, Ni2+. Autophosphorylation of insulin receptor was almost completely blocked by 10 microM Zn2+. Zn2+, however, did not appear to affect the binding of insulin to its receptor. Histidine, a chelator of Zn2+, protected against the inhibitory effects of Zn2+. The failure of histidine to regenerate the competence of the Zn2+-inhibited receptor to undergo autophosphorylation suggested that the inhibition by Zn2+ was irreversible. In addition to inhibiting autophosphorylation, Zn2+ inhibited the tyrosyl-protein kinase activity of highly purified phosphorylated receptor. Zn2+ was also observed to inhibit phosphotyrosyl-protein phosphatase activity present in preparations of partially purified insulin receptor. These inhibitory effects of Zn2+ should be considered in the design of protocols for the isolation and handling of insulin receptor and possibly other tyrosine kinases. Additionally, the possible physiological significance of the inhibition of insulin receptor kinase by Zn2+ is discussed in light of the fact that Zn2+ is accumulated in and secreted from pancreatic islet cells together with insulin.

Biological and biochemical characterization of a new isolate of feline sarcoma virus: Theilen-pedersen (TP1-FeSV)

A new feline sarcoma virus designated Theilen-Pedersen (TP1-FeSV) has been isolated from a spontaneous, slowly growing fibrosarcoma of a domestic short-haired 4-year-old castrated cat. The virus codes for a gag-onc fusion protein of 83,000 molecular weight phosphorylated in vivo at serine, threonine, and tyrosine residues. Cells transformed in vitro with TP1-FeSV exhibit five- to 10-fold elevated levels of phosphotyrosine over FeLV-infected cells. The gag-onc polyprotein has associated with it a tyrosyl protein kinase activity which in vitro results in autophosphorylation of the molecule at tyrosine residues. The fusion protein cannot be labeled metabolically with [ 3H]glucosamine and tunicamycin has no effect on the electrophoretic mobility of the in vivo [ 32P]orthophosphate-labeled fusion protein. The fusion protein, in common with the gag precursor Pr65 gag, can be metabolically labeled with palmitic acid.

Peptide growth factors

Interaction of growth factors with target cells involves the rapid internalization of ligand: receptor complexes. The receptor possesses intrinsic tyrosyl protein kinase activity which may, in part, mediate the biological activities of the peptide factors. Sequence data indicates that growth factors and/or their receptors are related to oncogene products some of which also possess tyrosyl kinase activity.

Properties and mechanism of action of viral and cellular tyrosyl protein kinases

We have demonstrated the usefulness of several angiotensin analogs as substrates for a number of tyrosyl protein kinases of viral or cellular origin. Results of initial rate studies with pp60 scr at varying substrate concentrations indicated that the mechanism was sequential; Michaelis constants for ATP and peptide were 7 μ m and 0.24 μ m respectively and V max was 1.0 nmol/min/mg. The end-product ADP and the ATP analog, AMP-PNP, were competitive inhibitors at varying ATP concentrations and noncompetitive inhibitors at varying peptide concentrations. A dead-end analog of angiotensin II, [ΔPhe 4]-angiotensin II, was a noncompetitive inhibitor at varying ATP concentrations but induced substrate inhibition at varying peptide concentrations. The kinetic data allowed us to conclude that the reaction proceeded via an Ordered Bi Bi mechanism with ATP as the first binding substrate. The available evidence allowed us to conclude that while pp60 src contained essential histidine and lysine residues in its active site, the kinase reaction does not involve a phosphoryl enzyme intermediate. Phosphorylation of the angiotensin peptides in vitro also has allowed us to demonstrate the presence of at least two tyrosyl protein kinases in the cytoplasm of normal rat liver cells. These kinases appear to be novel in that they are present in normal cells and are not stimulated by growth factors. Also, results of preliminary experiments indicate that these kinases are not immunologically related to the transforming gene products of Rous and Fujinami sarcoma viruses (unpublished observations). The identification of these new kinases represents another application of the angiotensin peptides as substrates for tyrosyl kinases (13). The results obtained do not exclude the possibility that there exist in rat liver other tyrosyl kinases that do not phosphorylate these particular peptide substrates. No attempt has been made to characterize tyrosyl kinases associated with the plasma membrane fraction. Although they repreent only a small fraction of the total activity of liver cells, the plasma membrane kinases have a relatively high specific activity. These kinases may be identical with growth factor receptor kinases previously identified in liver cell membranes (5). The most abundant tyrosyl kinase in rat liver cytoplasm has a molecular weight of 75,000 daltons and was found in cytosol and microsomal salt-wash fraction. The observation that the purified 75 Kd enzyme phosphorylates a 75 Kd protein on tyrosine residues suggests that the enzyme may possess autophosphorylating activity. A high molecular weight (greater than 160 Kd) kinase also was found in both the cytosol and microsomal salt-wash fraction. Since this high molecular weight material was very heterogeneous, we did not attempt to analyze its reaction products in an in vitro phosphorylation reaction. There is as yet no evidence to indicate whether the high molecular weight enzyme is an aggregated form of TPK 75 or whether it is an entirely different enzyme. Furthermore, it is not clear how these two kinases relate to those that were not extractable from microsomal membranes. Resolution of these issues will have to await further purification and characterization of the different enzymes. To summarize, rat liver cytoplasm may contain as many as three tyrosyl protein kinases: TPK 75, a high molecular weight enzyme, and an enzyme that associates tightly with microsomes. The available data do not allow us to conclude whether or not the association of tyrosyl kinases with ribosomes and endoplasmic reticulum is biologically significant. However, such an association is rather novel and suggests that tyrosyl kinases may play a role in regulating the activity of the protein synthetic machinery. Considerable evidence has been presented for the significance of phosphorylation of serine and threonine residues of ribosomal proteins and initiation factors in protein synthesis and cellular metabolic activities. Although there has not been any report of tyrosine-specific phosphorylation in these components, the involvement of phosphatases has not been adequately dealt with. In view of the findings presented in this report, these issues merit reevaluation.

Tyrosyl protein kinases in normal rat liver: identification and partial characterization.

Rat livers were fractionated and subcellular components were assayed for tyrosyl protein kinase activity. About 60% of the kinase activity in the cytoplasm sedimented with the microsomal fraction, whereas 40% remained in the supernatant. Purification of cytosolic and microsomal kinases by ion-exchange and gel filtration chromatography resolved a major species whose molecular mass was 75 kilodaltons (referred to as TPK 75) and a minor one whose molecular mass was greater than 160 kilodaltons. Partially purified TPK 75 phosphorylated a protein of the same molecular mass on tyrosine residues. The activity associated with TPK 75 was not stimulated by growth factors and was sensitive to thiol re-agents.

In vitro phosphorylation of angiotensin analogs by tyrosyl protein kinases.

Peptide analogs of angiotensin were phosphorylated in vitro by the src gene product, pp60src, of Rous sarcoma virus. The Km for the phosphorylation reaction varied from 1 to 5 mM and the Vmax varied from 2 to 10 nmol/min/mg. Tyrosine was the only residue phosphorylated in all analogs that were examined. The peptides were phosphorylated by tyrosyl protein kinases associated with several avian sarcoma viruses and by the epidermal growth factor-receptor kinase of A431 cells. Peptide substrate also was used to investigate the effectiveness of three different phosphatase inhibitors. Assay of tyrosyl kinase activities in whole cell lysates indicated that both p-nitrophenyl phosphate and sodium vanadate were potent inhibitors of phosphotyrosine phosphatases.

Association of the transforming proteins of Rous, Fujinami, and Y73 avian sarcoma viruses with the same two cellular proteins.

Two forms of the transforming proteins of Fujinami (pp140fps) and Yamaguchi 73 (pp94yes) sarcoma viruses were detected in lysates of chicken cells transformed by these viruses; the majority of pp140fps and pp94yes molecules were present as monomers; however, a small percentage of these proteins was associated in a complex with two cellular proteins of Mr 90,000 and 50,000. These cellular proteins were shown to be identical to those previously found to be complexed with the transforming protein of Rous sarcoma virus, pp60src. These results suggest a common role for the interaction of pp90 and pp50 with viral transforming proteins encoding tyrosyl-protein kinases.

Association of the transforming proteins of Rous, Fujinami, and Y73 avian sarcoma viruses with the same two cellular proteins.

Two forms of the transforming proteins of Fujinami (pp140fps) and Yamaguchi 73 (pp94yes) sarcoma viruses were detected in lysates of chicken cells transformed by these viruses; the majority of pp140fps and pp94yes molecules were present as monomers; however, a small percentage of these proteins was associated in a complex with two cellular proteins of Mr 90,000 and 50,000. These cellular proteins were shown to be identical to those previously found to be complexed with the transforming protein of Rous sarcoma virus, pp60src. These results suggest a common role for the interaction of pp90 and pp50 with viral transforming proteins encoding tyrosyl-protein kinases.

Tyrosylprotein kinase and phosphatase activities in membrane vesicles from normal and Rous sarcoma virus-transformed rat cells.

Membrane vesicles; isolated from normal and Rous sarcoma virus-transformed rat cells, have an associated cyclic-AMP independent kinase that phosphorylates a Mr 37,000 protein in vesicles from normal cells and proteins of Mr 37,000, 50,000, and 67,000 in vesicles from transformed cells. Proteins in vesicles from normal and transformed cells contain 9% and 77%, respectively, of their labeled phospho amino acids as phosphotyrosine. Thus, isolation of vesicles and subsequent incubation with [gamma-32P]ATP enriches the proportion of labeled phosphotyrosine in proteins (relative to other phospho amino acids) by two orders of magnitude over that found in intact cells. The in vitro phosphorylation of each of these proteins is enhanced in the presence of 10 microM Zn2+, a phosphotyrosylprotein phosphatase inhibitor. From these studies it appears that membrane vesicles may be a valuable system for examination of transformation-specific phosphorylation of proteins.