scholarly journals Protein kinase activity associated with stored messenger ribonucleoprotein particles of Xenopus oocytes.

1988 ◽  
Vol 107 (1) ◽  
pp. 45-56 ◽  
Author(s):  
A Cummings ◽  
J Sommerville
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Early Stage ◽  
Early Developmental Stage ◽  
Protein Kinase Activity ◽  
Mrna Accumulation ◽  
Messenger Ribonucleoprotein ◽  
Stored Mrna ◽  
Stage 1

As the oocytes of Xenopus laevis grow and develop they accumulate vast stores of mRNA for use during early embryogenesis. The stored mRNA is stabilized and may be prevented from being translated in oocytes by the binding of a defined set of oocyte-specific proteins to form messenger RNP (mRNP) particles. A key event in the interaction of protein with mRNA is the phosphorylation of those few polypeptides that bind directly to all classes of polyadenylated mRNA. In this study we show that the phosphorylating enzyme (protein kinase), in addition to its target phosphoproteins, is an integral component of the mRNP particles. This association extends through various stages in the formation and use of the mRNP particles. Examination of material from oocytes of an early developmental stage (early stage 1), when the level of accumulated mRNA is low, reveals an excess of protein particles free of RNA, sedimenting at 6-18 S, and containing protein kinase activity and mRNA-binding phosphoproteins. At stages of maximum rate of mRNA accumulation (stages 1 and 2), the phosphoproteins and kinase are found primarily in individual mRNP particles that sediment at 40-80 S. As ribosomes become abundant (stages 2 and 3), the mRNP particles tend to interact with ribosomal subunits, at least in vitro, to form blocked translation initiation complexes that sediment at 80-110 S. These results are compared with observation on stored mRNP in other developmental systems.

scholarly journals Novel Chemical Class of pUL97 Protein Kinase-Specific Inhibitors with Strong Anticytomegaloviral Activity

2004 ◽  
Vol 48 (11) ◽  
pp. 4154-4162 ◽  
Author(s):  
Thomas Herget ◽  
Martina Freitag ◽  
Monika Morbitzer ◽  
Regina Kupfer ◽  
Thomas Stamminger ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
High Efficiency ◽  
Fluorescent Protein ◽  
Human Fibroblasts ◽  
Growth Factor Receptor ◽  
Protein Kinase Activity ◽  
Hcmv Replication

ABSTRACT Human cytomegalovirus (HCMV) is a major human pathogen frequently associated with life-threatening disease in immunosuppressed patients and newborns. The HCMV UL97-encoded protein kinase (pUL97) represents an important determinant of viral replication. Recent studies demonstrated that pUL97-specific kinase inhibitors are powerful tools for the control of HCMV replication. We present evidence that three related quinazoline compounds are potent inhibitors of the pUL97 kinase activity and block in vitro substrate phosphorylation, with 50% inhibitory concentrations (IC50s) between 30 and 170 nM. Replication of HCMV in primary human fibroblasts was suppressed with a high efficiency. The IC50s of these three quinazoline compounds (2.4 ± 0.4, 3.4 ± 0.6, and 3.9 ± 1.1 μM, respectively) were in the range of the IC50 of ganciclovir (1.2 ± 0.2 μM), as determined by the HCMV green fluorescent protein-based antiviral assay. Importantly, the quinazolines were demonstrated to have strong inhibitory effects against clinical HCMV isolates, including ganciclovir- and cidofovir-resistant virus variants. Moreover, in contrast to ganciclovir, the formation of resistance to the quinazolines was not observed. The mechanisms of action of these compounds were confirmed by kinetic analyses with infected cells. Quinazolines specifically inhibited viral early-late protein synthesis but had no effects at other stages of the replication cycle, such as viral entry, consistent with a blockage of the pUL97 function. In contrast to epithelial growth factor receptor inhibitors, quinazolines affected HCMV replication even when they were added hours after virus adsorption. Thus, our findings indicate that quinazolines are highly efficient inhibitors of HCMV replication in vitro by targeting pUL97 protein kinase activity.

Structural and functional modification of pp60c-src associated with polyoma middle tumor antigen from infected or transformed cells

1985 ◽  
Vol 5 (10) ◽  
pp. 2647-2652
Author(s):  
C A Cartwright ◽  
M A Hutchinson ◽  
W Eckhart
Keyword(s):  
Protein Kinase ◽  
Tyrosine Phosphorylation ◽  
Tumor Antigen ◽  
Kinase Activity ◽  
Protein Kinase Activity ◽  
Amino Terminal ◽  
Exogenous Substrate ◽  
And Function ◽  
Amino Terminal Domain

The polyoma middle tumor antigen (MTAg) associates with the src proto-oncogene product pp60c-src in infected or transformed rodent cells. The tyrosine protein kinase activity of pp60c-src, as measured by in vitro phosphorylation of pp60c-src itself or the exogenous substrate enolase, was increased 10- to 20-fold in cells transformed or infected with transformation-competent polyoma virus compared with controls. pp60c-src associated with MTAg and precipitated with polyoma antitumor serum had a novel site(s) of in vitro tyrosine phosphorylation within its amino-terminal domain. These observations suggest that association of MTAg with pp60c-src alters the accessibility of pp60c-src tyrosine residues for phosphorylation in vitro and increases pp60c-src protein kinase activity. Several transformation-defective mutants of MTAg did not cause amino-terminal tyrosine phosphorylation of pp60c-src in vitro or enhance its protein kinase activity, suggesting that these properties correlate with the transforming ability of MTAg. However, one transformation-defective MTAg mutant, dl1015, did cause amino-terminal tyrosine phosphorylation of pp60c-src in vitro and did enhance its protein kinase activity. This suggests that properties of MTAg, in addition to modifying the structure and function of pp60c-src, may be important for transformation.

scholarly journals Expression of the mammalian c-fes protein in hematopoietic cells and identification of a distinct fes-related protein.

1985 ◽  
Vol 5 (10) ◽  
pp. 2543-2551 ◽  
Author(s):  
I MacDonald ◽  
J Levy ◽  
T Pawson
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Human Peripheral Blood ◽  
Hematopoietic Cells ◽  
Specific Protein ◽  
Protein Kinase Activity ◽  
Sarcoma Virus ◽  
Human And Mouse ◽  
Specific Protein Kinase

The avian c-fps and mammalian c-fes proto-oncogenes are cognate cellular sequences. Antiserum raised against the P140gag-fps transforming protein of Fujinami avian sarcoma virus specifically recognized a 92,000-Mr protein in human and mouse hematopoietic cells which was closely related in structure to Snyder-Theilen feline sarcoma virus P87gag-fes. This polypeptide was apparently the product of the human c-fes gene and was therefore designated p92c-fes. Human p92c-fes was associated with a tyrosine-specific protein kinase activity in vitro and was capable of both autophosphorylation and phosphorylation of enolase as an exogenous protein substrate. The synthesis of human and mouse p92c-fes was largely, though not entirely, confined to myeloid cells. p92c-fes was expressed to relatively high levels in a multipotential murine myeloid cell line, in more mature human and mouse granulocyte-macrophage progenitors, and in differentiated macrophage like cells as well as in the mononuclear fraction of normal and leukemic human peripheral blood. p92c-fes was not found in erythroid cells, with the exception of a human erythroleukemia line which retains the capacity to differentiate into macrophage like cells. These results suggest a normal role for the p92c-fes tyrosine kinase in hematopoiesis, particularly in granulocyte-macrophage differentiation. In addition, a distinct 94,000-Mr polypeptide, antigenically related to p92c-fes, was identified in a number of hematopoietic and nonhematopoietic human and mouse cells and was also found to be associated with a tyrosine-specific protein kinase activity.

Invariant phosphorylation of the Saccharomyces cerevisiae Cdc28 protein kinase

1988 ◽  
Vol 8 (7) ◽  
pp. 2976-2979
Author(s):  
J A Hadwiger ◽  
S I Reed
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Kinase Activity ◽  
Specific Protein ◽  
Protein Kinase Activity ◽  
Cycle Arrest ◽  
Phosphorylation Level ◽  
Proposed Regulation ◽  
Specific Protein Kinase

The phosphorylation level of the Saccharomyces cerevisiae Cdc28 protein remained invariant under conditions that resulted in cell cycle arrest in the G1 phase and loss of Cdc28-specific protein kinase activity when the activity was assayed in vitro. These results are in contrast to the proposed regulation of the homologous Cdc2 protein kinase of Schizosaccharomyces pombe.

scholarly journals Mapping of a self-interaction domain of the cytomegalovirus protein kinase pUL97

2007 ◽  
Vol 88 (2) ◽  
pp. 395-404 ◽  
Author(s):  
Vera Schregel ◽  
Sabrina Auerochs ◽  
Ramona Jochmann ◽  
Katja Maurer ◽  
Thomas Stamminger ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Kinase Domain ◽  
Protein Kinase Activity ◽  
Interaction Domain ◽  
Amino Acid Region ◽  
Substrate Phosphorylation ◽  
Regulatory Functions ◽  
Acid Region

The human cytomegalovirus-encoded protein kinase pUL97 is a determinant of efficient virus replication and fulfils several regulatory functions. In particular, pUL97 interacts with and phosphorylates viral and cellular proteins. Substrate phosphorylation has regulatory consequences on viral replicative stages such as DNA synthesis, transcription and nuclear capsid egress. pUL97, in accordance with related herpesviral protein kinases, possesses strong autophosphorylation activity. Here, we demonstrate that pUL97 shows a pronounced potential to self-interact. Self-interaction of pUL97 is not dependent on its kinase activity, as seen with a catalytically inactive point mutant. The property of self-interaction maps to the amino acid region 231–280 which is separable from the postulated kinase domain. The detection of high-molecular-mass complexes of pUL97 suggests the formation of dimers and oligomers. Importantly, the analysis of pUL97 mutants by in vitro kinase assays demonstrated a correlation between self-interaction and protein kinase activity, i.e. all mutants lacking the ability to self-interact were negative or reduced in their kinase activity. Thus, our findings provide novel insights into the pUL97 structure–activity relationship suggesting an importance of self-interaction for pUL97 functionality.

Differential protein kinase activity in ER-positive and ER-negative breast cancer

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. e22142-e22142
Author(s):  
R. Ruijtenbeek ◽  
A. Umar ◽  
L. van Houten ◽  
R. Hilhorst ◽  
J. A. Foekens ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Protein Kinase ◽  
Kinase Activity ◽  
Breast Tumors ◽  
Protein Kinase Activity ◽  
Vitro Assay ◽  
Peptide Substrates ◽  
Peptide Microarrays ◽  
Er Positive

e22142 Background: Specific protein tyrosine kinases (PTKs) promote cancer progression and are potential drug targets. The presence of the estrogen receptor (ER) is an important criterion in deciding on treatment of patients with breast cancer. Since PTKs can affect the tumors' receptor function, we investigated in our first proof-of-principle study whether it is possible to discriminate ER-positive (ER+) from ER-negative (ER-) tumors based on PTK activity using microarrays containing many PTK peptide substrates. Methods: Cryosections of 12 ER+ and 12 ER- breast tumors were lysed in the presence of appropriate protease and phosphatase inhibitors. The protein kinase activity in the lysates was monitored in vitro using PamChip peptide microarrays, which comprise of 253 PTK peptide substrates derived from known human phosphorylation sites. Peptide phosphorylation through active kinases can be monitored in samples in real time, using a fluorescently labeled phospho-tyrosine specific antibody. Results: Phosphorylation activity profiles were determined in quadruplicate using multiple independent sample preparations. Using ANOVA analysis, out of 253 peptides, several peptides with a different phosphorylation signal in ER+ and ER- samples were found: 22 with p < 0.02 including VEGFR1, 2 and 3 and ADAM9. Multivariate unsupervised analysis using Principle Component Analysis showed a clustering of ER+ samples vs. ER- samples. Predictive analysis was performed using Partial-Least Squares Discriminant Analysis without explicit feature selection. The prediction error obtained from within experiment cross validation was typically in the range 10–20%. The error rate for between experiment prediction was 20%. An average predictive profile was constructed in which peptides with a relatively large weight were included, e.g. ADAM9 and BCAR1. Conclusions: Using PamChip peptide microarrays we have shown that differences in protein kinase activity exist between ER+ and ER- breast tumors. Our in vitro assay is a promising tool to investigate the interplay between kinase and nuclear receptor mediated signaling, with potential relevance to patient selection for targeted therapies. No significant financial relationships to disclose.

scholarly journals Cks1 Is Required for G1Cyclin–Cyclin-Dependent Kinase Activity in Budding Yeast

2000 ◽  
Vol 20 (16) ◽  
pp. 5858-5864 ◽  
Author(s):  
Gregory J. Reynard ◽  
William Reynolds ◽  
Rati Verma ◽  
Raymond J. Deshaies
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Budding Yeast ◽  
Protein Kinase Activity ◽  
Cyclin Dependent Kinase ◽  
Multiple Substrates ◽  
Cell Extracts

ABSTRACT p13suc1 (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 G1 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 G1cyclin-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 G1 cyclins, which depends on Cdc28 activity, is reduced in cks1-38 cells. The role of Cks1 in promoting G1 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 G1 phase in budding yeast.

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