Phosphorylation Of Tyrosyl Residues Research Articles

Regulation of phospholipase D by muscarinic receptors in rat submandibular ductal cells

The muscarinic agonist carbachol stimulated phospholipase D (PLD) in rat submandibular gland (RSMG) ductal cells in a time and concentration-dependent manner. This effect was inhibited by chelation of extracellular calcium with ethylene glycol-bis(β-aminoethyl ether)- N, N, N′, N′-tetraacetic acid (EGTA). PLD could also be activated by epinephrine and AlF 4 −, two polyphosphoinositide-specific phospholipase C (PPI-PLC) activators, and by the phorbol ester o-tetradecanoylphorbol 13-acetate (TPA) which activates protein kinase C (PKC). Ionomycin and thapsigargin only slightly increased PLD activity. Ortho-vanadate, a tyrosine phosphatase inhibitor, also stimulated PLD activity. Both carbachol and o-vanadate increased the formation of inositol phosphates and the tyrosine phosphorylation of at least two proteins (55–60 and 120 kDa). Calphostin C (a PKC inhibitor), U73122 (a PPI-PLC inhibitor) and genistein (a tyrosine kinase inhibitor) blocked the activation of PLD, of PLC and the phosphorylation of tyrosyl residues in response to carbachol and vanadate. Taken together, these results suggest that rat submandibular gland ductal cells express a calcium-dependent PLD activity. This enzyme is regulated by carbachol via a PLC–PKC–tyrosine kinase pathway.

Threonine Phosphorylation of the β3 Integrin Cytoplasmic Tail, at a Site Recognized by PDK1 and Akt/PKB in Vitro, Regulates Shc Binding

The mechanism of outside-in signaling by integrins parallels that for growth factor receptors. In both pathways, phosphorylation of a cytoplasmic segment on tyrosine generates a docking site for proteins containing Src homology 2 (SH2) and phosphotyrosine binding domains. We recently observed that phosphorylation of a threonine (Thr-753), six amino acids proximal to tyrosine 759 in beta(3) of the platelet specific integrin alpha(IIb)beta(3), inhibits outside-in signaling through this receptor. We hypothesized that the presence of phosphothreonine 753 either renders beta(3) a poor substrate for tyrosine kinases or inhibits the docking capabilities of the tyrosyl-phosphorylated form of beta(3.) The first alternative was tested by comparing the phosphorylation of beta(3) model peptides by the tyrosine kinase pp60(c-src) and we found that the presence of a phosphate group on a residue corresponding to Thr-753 did not detectably alter the kinetics of tyrosine phosphorylation. However, the presence of phosphate on this threonine inhibited the binding of Shc to tyrosyl-phosphorylated beta(3) peptide. The inhibitory effect of the phosphate group could be mimicked by substituting an aspartic acid for Thr-753, suggesting that a negative charge at this position modulates the binding of Shc and possibly other phosphotyrosine binding domain- and SH2-containing proteins. A survey of several protein kinases revealed that Thr-753 was avidly phosphorylated by PDK1 and Akt/PKB in vitro. These observations suggest that activation of PDK1 and/or Akt/PKB in platelets may modulate the binding activity and/or specificity of beta(3) for signaling molecules.

The β-Adrenergic Receptor Is a Substrate for the Insulin Receptor Tyrosine Kinase

G-protein-linked receptors and intrinsic tyrosine-kinase growth receptors represent two prominent modalities in cell signaling. Cross-regulation among members of both receptor superfamilies has been reported, including the counter-regulatory effects of insulin on beta-adrenergic catecholamine action. Cells stimulated by insulin show loss of function and increased phosphotyrosine content of beta 2-adrenergic receptors. Phosphorylation of tyrosyl residues 350/354 of beta 2-adrenergic receptors is obligatory for counter-regulation by insulin (Karoor, V., Baltensperger, K., Paul, H., Czech, M., and Malbon, C. C. (1995) J. Biol. Chem. 270, 25305-25308), suggesting the hypothesis that G-protein-linked receptors themselves may act as substrates for the insulin receptor and other growth factor receptors. This hypothesis was evaluated directly using recombinant human insulin receptor, hamster beta 2-adrenergic receptor, and an vitro reconstitution and phosphorylation assay. Insulin is shown to stimulate insulin receptor-catalyzed phosphorylation of the beta 2-adrenergic receptor. Phosphoamino acid analysis establishes that insulin receptor-catalyzed phosphorylation of the beta 2-adrenergic receptor in vitro is confined to phosphotyrosine. High pressure liquid chromatography and two-dimensional mapping reveal insulin receptor-catalyzed phosphorylation of the beta 2-adrenergic receptor at residues Tyr132/Tyr141, Tyr350/Tyr354, and Tyr364, known sites of phosphorylation in response to insulin in vivo. Insulin-like growth factor-I receptor as well as the insulin receptor displays the capacity to phosphorylate the beta 2-adrenergic receptor in vitro, establishing a new paradigm, i.e. G-protein-linked receptors acting as substrates for intrinsic tyrosine kinase growth factor receptors.

Possible Role of Tyrosine Kinases in the Prolactin Stimulation of Cell Division in Nb2Node Lymphoma Cells

Orthovanadate and genistein were employed to determine the possible role of tyrosine kinase in the prolactin stimulation of cell division in Nb2 node lymphoma cells. Orthovanadate (0.1-0.25 microM), an inhibitor of specific phosphatases, was found to stimulate cell division as well as potentiate the mitogenic effect of prolactin on Nb2 cells when prolactin was employed at a less than maximum stimulatory concentration. Genistein, an inhibitor of tyrosine kinase, was found to inhibit the prolactin stimulation of cell division. Since prolactin was shown in earlier studies to stimulate the phosphorylation of tyrosyl residues in specific Nb2 cell proteins, these results suggest that the signal transduction pathway for the prolactin stimulation of cell division in the Nb2 node lymphoma cells likely involves the participation of tyrosine kinase(s).

Structure, biosynthesis, and transduction properties of the human mu-psi L complex: similar behavior of preB and intermediate preB-B cells in transducing ability.

In human preB cells, the mu chain is associated with a surrogate light chain composed of the VpreB and lambda-like gene products. Using anti-peptide antibodies directed against VpreB and lambda-like epitopes, we identified the discrete components of the mu-psi L (pseudo-light) chain complex in various preB cell lines, and in intermediate preB-B cells that co-expressed the psi L and the kappa chain. The lambda-like gene product was identified as a single band at 20 kDa, disulfide linked to the mu chain. VpreB was detected at 16 kDa and, depending upon the cell lines, an isoform of this polypeptide was also present at 15 kDa. In addition, lambda-like--VpreB chain complexes not associated with mu were identified both in cell lysates and culture supernatants. Pulse-chase experiments indicated that VpreB was transiently associated with two new polypeptides of molecular weights 17.5 and 36 kDa. Expression of mu-psi L and co-expression of mu-psi L and microL at the surface of preB and intermediate preB-B cells respectively was detected by cytofluorimetry. The signal transduction ability of the complex in both types of cells was shown by measuring the calcium mobilization and the phosphorylation of tyrosyl residues upon stimulation by anti-mu. Signal events were similar in both cases, but differed from those induced in a mature B cell line. This points to a definite function of the preB cell receptor and suggests that the intermediate preB-B cell line still lacks some molecular components that condition initiation of a mature B cell transduction signal.

Structure, Regulation, and Function of Protein Tyrosine Phosphatases

The phosphorylation of tyrosyl residues in proteins is an essential aspect of many signal transduction events, including the control of both normal and neoplastic cell growth and proliferation. Since the earliest observations of tyrosine phosphorylation, it has been appreciated that this is a reversible process in which the net level of phosphate in a target substrate reflects the balance between the competing action of kinases and phosphatases. Thus, in cells transformed by temperature-sensitive mutants of Rous sarcoma virus, an elevation in the levels of phosphotyrosine is observed at the permissive temperature, at which the kinase is active; however, if the cells are shifted to the nonpermissive temperature, at which the kinase is inactivated, a rapid dephosphorylation of tyrosyl residues ensues due to the action of protein tyrosine phosphatases (PTPases) (Sefton et al. 1980). The last 10 years has witnessed great progress in the characterization of the protein tyrosine kinases, whereas...

Effect of Microinjection of a Low-Mr Human Placenta Protein Tyrosine Phosphatase on Induction of Meiotic Cell Division in Xenopus Oocytes

Homogeneous preparations of a protein phosphatase that is specific for phosphotyrosyl residues (protein tyrosine phosphatase [PTPase] 1B) were isolated from human placenta and microinjected into Xenopus oocytes. This resulted in an increase in activity of up to 10-fold over control levels, as measured in homogenates with use of an artificial substrate (reduced carboxamidomethylated and maleylated lysozyme). Microinjected PTPase was stable for at least 18 h. It is distributed within the oocyte in a manner similar to the endogenous activity and is suggestive of an interaction with cellular structures or molecules located predominantly in the animal hemisphere. The phosphatase markedly retarded (by up to 5 h) maturation induced by insulin. This, in conjunction with the demonstration that PTPase 1B abolished insulin stimulation of an S6 peptide (RRLSSLRA) kinase concomitant with a decrease in the phosphorylation of tyrosyl residues in a protein with the same apparent Mr as the beta subunit of the insulin and insulinlike growth factor 1 receptors (M. F. Cicirelli, N. K. Tonks, C. D. Diltz, E. H. Fischer, and E. G. Krebs, submitted for publication), provides further support for an essential role of protein tyrosine phosphorylation in insulin action. Furthermore, maturation was significantly retarded even when the PTPase was injected 2 to 4 h after exposure of the cells to insulin. PTPase 1B also retarded maturation induced by progesterone and maturation-promoting factor, which presumably do not act through the insulin receptor. These data point to a second site of action of the PTPase in the pathway of meiotic cell division, downstream of the insulin receptor and following the appearance of active maturation-promoting factor.

Effect of microinjection of a low-Mr human placenta protein tyrosine phosphatase on induction of meiotic cell division in Xenopus oocytes.

Homogeneous preparations of a protein phosphatase that is specific for phosphotyrosyl residues (protein tyrosine phosphatase [PTPase] 1B) were isolated from human placenta and microinjected into Xenopus oocytes. This resulted in an increase in activity of up to 10-fold over control levels, as measured in homogenates with use of an artificial substrate (reduced carboxamidomethylated and maleylated lysozyme). Microinjected PTPase was stable for at least 18 h. It is distributed within the oocyte in a manner similar to the endogenous activity and is suggestive of an interaction with cellular structures or molecules located predominantly in the animal hemisphere. The phosphatase markedly retarded (by up to 5 h) maturation induced by insulin. This, in conjunction with the demonstration that PTPase 1B abolished insulin stimulation of an S6 peptide (RRLSSLRA) kinase concomitant with a decrease in the phosphorylation of tyrosyl residues in a protein with the same apparent Mr as the beta subunit of the insulin and insulinlike growth factor 1 receptors (M. F. Cicirelli, N. K. Tonks, C. D. Diltz, E. H. Fischer, and E. G. Krebs, submitted for publication), provides further support for an essential role of protein tyrosine phosphorylation in insulin action. Furthermore, maturation was significantly retarded even when the PTPase was injected 2 to 4 h after exposure of the cells to insulin. PTPase 1B also retarded maturation induced by progesterone and maturation-promoting factor, which presumably do not act through the insulin receptor. These data point to a second site of action of the PTPase in the pathway of meiotic cell division, downstream of the insulin receptor and following the appearance of active maturation-promoting factor.

Phosphorylation of protein tyrosine by human erythrocyte casein kinase A

Human erythrocyte casein kinase A, previously identified as a seryl, threonyl kinase, was found also to catalyze the phosphorylation of protein tyrosine. Phosphorylation of tyrosyl residues was detected in angiotensin-II and in several tyrosine containing synthetic peptides. In addition, phosphorylation on tyrosyl residues was also observed in alkylated bovine serum albumin and in band 3 and ankyrin purified from human erythrocyte membranes. The identification of phosphotyrosine was conducted using two-dimensional thin layer electrophoresis at pH 1.9 and 3.5 after acid hydrolysis of the phosphoproteins. It should be noted, however, that the major phosphorylation sites in band 3 and ankyrin catalyzed by casein kinase A were seryl and threonyl residues.