Presence Of Tyrosine Kinase Activity Research Articles

Detection of histidine kinases via a filter-based assay and reverse-phase thin-layer chromatographic phosphoamino acid analysis

The methods that detect histidine phosphorylation have largely been either laborious or difficult to apply quantitatively. The major difficulty in assessing for its presence is its alkali-stable, acid-labile nature. While an assay that detects alkali-stable phosphorylation has been developed, it does not distinguish phosphohistidine from other alkali-stable phosphoamino acids. Using this established method, we extend the assay to facilitate the specific detection of phosphohistidine. We use the acid-lability of phosphohistidine as a defining feature in our approach for its detection. In addition, reverse-phase thin-layer chromatography was utilized to conclusively demonstrate the viability of the conditions that we implement in the assay for the selective detection of phosphohistidine. In summary, this report describes a rapid filter-based kinase assay that quantitatively measures histidine kinase activity, even in the presence of tyrosine kinase activity.

Phosphorylation of plant proteins and the identification of protein-tyrosine kinase activity in maize seedlings.

Phosphotyrosine was found to be 0.5% of the total phosphoamino acids labelled with [32P]orthophosphate in endogenous maize seedlings proteins. Two peaks of protein kinase activity towards phosphorylation of synthetic peptide poly (Glu80, Tyr20) were obtained after chromatography of protein extract of dark-grown etiolated maize seedlings on phosphocellulose. The phosphorylation of synthetic peptide as well as endogenous proteins was strongly stimulated by Mn2+. At least three endogenous proteins with molecular masses in the range of 40-65 kDa were predominantly phosphorylated. This phosphorylation was resistant to alkali treatment. Chemical, immunological and enzymatic data indicated the presence of tyrosine kinase activity and also phosphotyrosine in proteins of maize seedlings. The plant enzyme(s) is reminiscent known mammalian cytosolic tyrosine kinase(s).

Detection of sarcoma virus family tyrosine kinase activity in coronary arterial tissue.

We have observed that the tyrosine kinase inhibitors genistein and tyrphostin can selectively block angiotensin II mediated and vasopressin-mediated contractions in porcine coronary arterial strips, without affecting the action of acetylcholine. Therefore, we assessed the presence of tyrosine kinase activity in the porcine coronary artery tissue, using an assay specific for sarcoma virus (src) related tyrosine kinases. In both membrane and cytosolic fractions of porcine coronary artery, we detected src-related tyrosine kinase activity that could be inhibited by both genistein and tyrphostin. The tyrosine kinase activity in membrane extracts was separated into two peaks by sequential chromatography on hydroxylapatite and Mono-Q columns. Protein in both peaks exhibited Western blot cross-reactivity with anti-src antibodies and contained tyrosine kinase activity that was inhibited by genistein and tyrphostin. We conclude that porcine coronary artery tissue contains src-related tyrosine kinase activity. However, because of the comparatively low sensitivity of the isolated src kinase activity towards genistein and tyrphostin, compared with the much higher sensitivity of the contractile response to these inhibitors, a direct role for c-rsc in the regulation of contractions elicited by agonists such as angiotensin II and arginine vasopressin cannot yet be assigned.

Phosphorylation of Tyrosine Residues of RNA Polymerase II and Other Nuclear Proteins by Active Chromatin Tyrosin Kinase(s)

We demonstrate here for the first time that protein tyrosine kinases are present in the active chromatin of nucleus. The presence of tyrosine kinase activity in the active chromatin was initially determined using poly (Glu,Na-Tyr;4:1) (PGT) as a tyrosine phosphorylatable substrate. Active chromatin in the presence of cofactors phosphorylated PGT at a rate of 40 pmol/min. The phosphorylation of PGT by active chromatin was inhibited by 41, 47 and ∼95% with genistein, n-ethylmaleimide and quercetin (known inhibitors of tyrosine kinases), respectively. A Lineweaver-Burk plot revealed an apparent Km of 50 μg/ml and Vmax of 45 pmol/min for active chromatin tyrosine kinase(s). Analyses of phosphorylation of endogenous substrates by immunoprecipitation, western blotting and phosphoamino acids revealed that active chromatin protein tyrosine kinase(s) are able to phosphorylate tyrosine residues of the large subunit of RNA pol II and several other active chromatin proteins. The ability of AC-PTKs to phosphorylate many proteins of active chromatin components argues strongly for its role(s) in regulating transcription.

Characterization of an interleukin-2 (IL-2)-induced tyrosine phosphorylated 116-kDa protein associated with the IL-2 receptor beta-subunit.

In this paper we have extended previous results on interleukin-2 receptor (IL2-R) signal transduction and focused on the interleukin-2 (IL-2)-stimulated tyrosine phosphorylation of a 116-kDa protein (p116) observed in IL-2 responsive cells. This protein exhibited rapid and transient phosphorylation kinetics in both human T-lymphocytes and the YT cell line, attaining maximum tyrosine phosphorylation within 5 min of stimulation with IL-2. Tyrosine phosphorylated p116 co-purified with activated IL-2 receptor beta-chain (IL2-R beta) when IL2-R complexes were covalently stabilized with the membrane-permeable cleavable cross-linking agent dithiobis(succimidyl propionate) prior to detergent cell lysis and immunoprecipitation with monoclonal anti-IL2-R beta antibodies. Under these conditions comparable amounts of tyrosine-phosphorylated p116 were immunoprecipitated with either anti-IL2-R beta antibodies or anti-phosphotyrosine antibodies, suggesting that a major portion of tyrosine phosphorylated p116 is associated with the IL2-R beta subunit. Furthermore, unphosphorylated p116 was also associated with unactivated IL2-R beta, based on the observation that p116 from unstimulated YT cells underwent tyrosine phosphorylation in IL2-R beta immune-complex tyrosine kinase assay as demonstrated by anti-phosphotyrosine immunoblotting. The presence of tyrosine kinase activity in affinity-purified IL2-R beta complexes supports the notion of a preformed receptor-kinase complex. The co-association of both p116 and tyrosine kinase activity with the IL2-R beta supports the critical role of the beta-chain in IL2-R signal transduction and suggests that p116 may have a role in the dynamics of IL2-R activation.

Demonstration of Growth Hormone (GH) Receptor- Associated Tyrosine Kinase Activity in Multiple GHResponsive Cell Types*

Highly purified GH-receptor preparations from 3T3-F442A fibroblasts, whose differentiation into adipocytes is promoted by GH, have been shown to contain a tyrosine kinase capable of phosphorylating GH receptors. In the current work, characteristics of the tyrosine kinase responsible for the in vitro phosphorylation of GH receptors from cultured 3T3-F442A fibroblasts were examined, and the presence of this GH receptor-associated tyrosine kinase activity was demonstrated in multiple cell types. GH-receptor complexes from GH-treated cells were partially purified by immunoprecipitation using anti-GH antibodies and then incubated as an immune complex with [gamma 32P] ATP. Incorporation of 32P into the GH receptor from 3T3-F442A fibroblasts was apparent within 1 min at 30 C after the addition of [gamma 32P]ATP (5-10 microM). A divalent cation was requisite for the phosphorylation; Mn2+ was significantly more effective than Mg2+ and Co2+; Ba2+, Ca2+, or Zn2+ had no effect. Excess unlabeled ATP, but not cytosine triphosphate, GTP, or uridine triphosphate, abolished 32P incorporation into the GH receptor and [gamma 32P]GTP could not replace [gamma 32P]ATP as a source of 32P. At 5.5 mM Mn2+, phosphorylation exhibited a biphasic dose response to ATP, with maximal phosphorylation occurring at a concentration of 10 microM ATP. At more physiological concentrations of ATP (1 mM), phosphorylation of the GH receptor was also stimulated by lower concentrations of Mn2+ (as low as 500 nM). Optimal reaction conditions determined for the phosphorylation reaction in 3T3-F442A fibroblasts were used to demonstrate incorporation of 32P from [gamma 32P]ATP into partially purified GH receptors from cultured human IM-9 lymphocytes, murine 3T3-F442A adipocytes, rat H-35 hepatoma cells, and freshly isolated rat adipocytes. The 32P was shown to be incorporated into tyrosyl residues in receptors from the two cell types tested (IM-9 lymphocytes and rat adipocytes). Cross-linked [125I] hGH-receptor complexes solubilized from the four cell types (IM-9 lymphocytes, 3T3-F442A adipocytes, H-35 hepatoma cells, and freshly isolated rat adipocytes) bound to and could be eluted from phosphotyrosyl antibody, suggesting that tyrosyl phosphorylation of GH receptors in all of these cells occurs in vivo. The presence of tyrosine kinase activity associated with GH receptors in multiple cell types from different species is consistent with tyrosine kinase activity playing a role in the actions of GH.

Protein tyrosine kinase activity and its substrates in rat intestinal microvillus membranes

Tyrosine phosphorylation has recently been recognized as a unique system involved in the regulation of cellular growth and differentiation. The role of tyrosine kinase activity in regulating intestinal proliferation has received little attention. The aim of this study was to document the presence of tyrosine kinase activity in intestinal microvillus membranes and to characterize the major endogenous tyrosine kinase substrates in microvillus membranes. Microvillus membranes, prepared from 21-day gestation fetal and adult CD rats by the calcium precipitation method, were solubilized in 0.1% Triton X-100 and incubated with [32P]adenosine triphosphate and (Glu80Tyr20)n, which is a heterogeneous population of synthetic peptides containing glutamate and tyrosine. Fetal, and to a lesser extent adult, microvillus membranes were shown to phosphorylate (Glu80Tyr20)n assayed by trichloroacetic acid-precipitable 32P incorporation as well as autoradiography of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Preliminary identification of the endogenous substrates of microvillus membrane tyrosine kinase activity was determined by three techniques. First, phosphorylated microvillus membrane proteins were solubilized in sodium dodecyl sulfate and separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the resultant gel was incubated in 1 mol/L KOH at 55 °C to selectively retain phosphotyrosine proteins. The patterns of tyrosine phosphorylation were dissimiliar in fetal and adult microvillus membranes; specifically, two major bands, 80 and 190 kilodaltons, were present in fetal microvillus membranes and were not prominent in adult microvillus membranes. These proteins were both phosphorylated on tyrosine residues as determined by phosphoamino acid analysis. Second, a specific antiphosphotyrosine monoclonal antibody was used to immunoprecipitate phosphotyrosine proteins from solubilized phosphorylated microvillus membranes. This antibody specifically immunoprecipitated proteins of molecular weights 36 and 68 from fetal and 33, 54, and 68 from adult microvillus membranes. Third, using a polyclonal antiphosphotyrosine antibody, Western blot analysis showed that the 68-kilodalton protein is the most abundant phosphotyrosine protein in fetal and adult microvillus membranes. These data will focus new investigations into the cellular mechanisms of the regulation of intestinal growth, particularly the role of luminal factors that may modulate microvillus membrane tyrosine kinase, and thus modulate enterocyte proliferation, differentiation, and function.