scholarly journals Requirement of phosphatidylinositol-3 kinase modification for its association with p60src.

1991 ◽  
Vol 11 (4) ◽  
pp. 1972-1979 ◽  
Author(s):  
Y Fukui ◽  
H Hanafusa
Keyword(s):  
Protein Kinase ◽  
Tyrosine Phosphorylation ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Sh2 Domain ◽  
Protein Kinase Activity ◽  
Phosphatidylinositol 3 Kinase ◽  
Tyrosine Residues ◽  
Phosphorylated Form ◽  
Chicken Embryo Fibroblasts

When purified p60v-src was mixed with lysates of chicken embryo fibroblasts and immunoprecipitated with anti-Src antibody, phosphatidylinositol (PI)-3 kinase activity was found to be present in the Src protein immunoprecipitates. The level of bound PI-3 kinase activity was 5 to 10 times higher in lysates obtained from cells transformed by the src, fps, or yes oncogene than in lysates of uninfected cells. This increase in associated PI-3 kinase activity appears to be due to increased binding of this enzyme to p60v-src. This change most likely resulted from tyrosine phosphorylation of PI-3 kinase or an associated protein, since the PI-3 kinase activity that can bind to p60v-src was depleted by antiphosphotyrosine antibody. Binding of PI-3 kinase did not require either p60src protein kinase activity or autophosphorylation of p60v-src tyrosine residues. Furthermore, binding was markedly decreased by deletions in the N-terminal SH2 region but unchanged by deletion of the C-terminal half of p60v-src containing the catalytic domain. Taking these data together, it appears that PI-3 kinase or its associated protein is phosphorylated on tyrosine and that the phosphorylated form can bind to the N-terminal half of p60v-src, which contains the SH2 domain.

Requirement of phosphatidylinositol-3 kinase modification for its association with p60src

1991 ◽  
Vol 11 (4) ◽  
pp. 1972-1979
Author(s):  
Y Fukui ◽  
H Hanafusa
Keyword(s):  
Protein Kinase ◽  
Tyrosine Phosphorylation ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Sh2 Domain ◽  
Protein Kinase Activity ◽  
Phosphatidylinositol 3 Kinase ◽  
Tyrosine Residues ◽  
Phosphorylated Form ◽  
Chicken Embryo Fibroblasts

When purified p60v-src was mixed with lysates of chicken embryo fibroblasts and immunoprecipitated with anti-Src antibody, phosphatidylinositol (PI)-3 kinase activity was found to be present in the Src protein immunoprecipitates. The level of bound PI-3 kinase activity was 5 to 10 times higher in lysates obtained from cells transformed by the src, fps, or yes oncogene than in lysates of uninfected cells. This increase in associated PI-3 kinase activity appears to be due to increased binding of this enzyme to p60v-src. This change most likely resulted from tyrosine phosphorylation of PI-3 kinase or an associated protein, since the PI-3 kinase activity that can bind to p60v-src was depleted by antiphosphotyrosine antibody. Binding of PI-3 kinase did not require either p60src protein kinase activity or autophosphorylation of p60v-src tyrosine residues. Furthermore, binding was markedly decreased by deletions in the N-terminal SH2 region but unchanged by deletion of the C-terminal half of p60v-src containing the catalytic domain. Taking these data together, it appears that PI-3 kinase or its associated protein is phosphorylated on tyrosine and that the phosphorylated form can bind to the N-terminal half of p60v-src, which contains the SH2 domain.

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 Enzyme activity effects of N-terminal His-tag attached to catalytic sub-unit of phosphoinositide-3-kinase

2013 ◽  
Vol 33 (6) ◽  
Author(s):  
James M. J. Dickson ◽  
Woo-Jeong Lee ◽  
Peter R. Shepherd ◽  
Christina M. Buchanan
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Cell Signalling ◽  
Protein Kinase Activity ◽  
Lipid Kinase ◽  
His Tag ◽  
The Impact

NTT (N-terminal tags) on the catalytic (p110) sub-unit of PI 3-K (phosphoinositol 3-kinase) have previously been shown to increase cell signalling and oncogenic transformation. Here we test the impact of an NT (N-terminal) His-tag on in vitro lipid and protein kinase activity of all class-1 PI 3-K isoforms and two representative oncogenic mutant forms (E545K and H1047R), in order to elucidate the mechanisms behind this elevated signalling and transformation observed in vivo. Our results show that an NT His-tag has no impact on lipid kinase activity as measured by enzyme titration, kinetics and inhibitor susceptibility. Conversely, the NT His-tag did result in a differential effect on protein kinase activity, further potentiating the elevated protein kinase activity of both the helical domain and catalytic domain oncogenic mutants with relation to p110 phosphorylation. All other isoforms also showed elevated p110 phosphorylation (although not statistically significant). We conclude that the previously reported increase in cell signalling and oncogenic-like transformation in response to p110 NTT is not mediated via an increase in the lipid kinase activity of PI 3-K, but may be mediated by increased p110 autophosphorylation and/or other, as yet unidentified, intracellular protein/protein interactions. We further observe that tagged recombinant protein is suitable for use in in vitro lipid kinase screens to identify PI 3-K inhibitors; however, we recommend that in vivo (including intracellular) experiments and investigations into the protein kinase activity of PI 3-K should be conducted with untagged constructs.

scholarly journals Biological and biochemical properties of the c-src+ gene product overexpressed in chicken embryo fibroblasts.

1989 ◽  
Vol 9 (8) ◽  
pp. 3332-3341 ◽  
Author(s):  
J B Levy ◽  
J S Brugge
Keyword(s):  
Protein Kinase ◽  
Chicken Embryo ◽  
Kinase Activity ◽  
Biochemical Properties ◽  
Serine Phosphorylation ◽  
Protein Kinase Activity ◽  
Independent Manner ◽  
Amino Terminal ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts

The c-src protein isolated from neuronal cells (pp60c-src+) displays a higher level of protein kinase activity than does pp60c-src from nonneural tissues. There are two structural alterations present in the amino-terminal half of pp60c-src+ expressed in neurons which could contribute to the enhanced activity of this form of pp60c-src: (i) a hexapeptide insert located at amino acid 114 of avian pp60c-src+ and (ii) a novel site(s) of serine phosphorylation. We characterized pp60c-src+ expressed in a nonneuronal cell type to identify factors that regulate the activity of the c-src+ protein and the importance of the neuronal environment on this regulation. The c-src+ protein overexpressed in chicken embryo fibroblasts (CEFs) displayed higher kinase activity than did pp60c-src. The major sites of phosphorylation of the c-src+ protein were Ser-17 and Tyr-527. The unique site(s) of serine phosphorylation originally identified in pp60c-src+ expressed in neurons was not detected in the c-src+ protein overexpressed in CEFs. Therefore, the hexapeptide insert is sufficient to cause an elevation in the tyrosine protein kinase activity of pp60c-src+. Our data also indicate that CEFs infected with the Rous sarcoma virus (RSV)c-src+ display phenotypic changes that distinguish them from cultures producing pp60c-src and that pp60c-src+-expressing cells are better able to grow in an anchorage-independent manner. The level of total cellular tyrosine phosphorylation in RSVc-src+-infected cultures was moderately higher than the level observed in cultures infected with RSVc-src. This level was not as pronounced as that observed in cells infected with RSVv-src or oncogenic variants of RSVc-src. Thus, pp60c-src+ could be considered a partially activated c-src variant protein much like other c-src proteins that contain mutations in the amino-terminal domain.

scholarly journals Sumoylation of AMPKβ2 subunit enhances AMP-activated protein kinase activity

2013 ◽  
Vol 24 (11) ◽  
pp. 1801-1811 ◽  
Author(s):  
Teresa Rubio ◽  
Santiago Vernia ◽  
Pascual Sanz
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Protein Kinase Activity ◽  
Energy Status ◽  
Regulatory Subunits ◽  
Cellular Energy ◽  
Sumo Ligase ◽  
Ampk Activity ◽  
Amp Activated Protein Kinase

AMP-activated protein kinase (AMPK) is a sensor of cellular energy status. It is a heterotrimer composed of a catalytic α and two regulatory subunits (β and γ). AMPK activity is regulated allosterically by AMP and by the phosphorylation of residue Thr-172 within the catalytic domain of the AMPKα subunit by upstream kinases. We present evidence that the AMPKβ2 subunit may be posttranslationally modified by sumoylation. This process is carried out by the E3-small ubiquitin-like modifier (SUMO) ligase protein inhibitor of activated STAT PIASy, which modifies the AMPKβ2 subunit by the attachment of SUMO2 but not SUMO1 moieties. Of interest, AMPKβ1 is not a substrate for this modification. We also demonstrate that sumoylation of AMPKβ2 enhances the activity of the trimeric α2β2γ1 AMPK complex. In addition, our results indicate that sumoylation is antagonist and competes with the ubiquitination of the AMPKβ2 subunit. This adds a new layer of complexity to the regulation of the activity of the AMPK complex, since conditions that promote ubiquitination result in inactivation, whereas those that promote sumoylation result in the activation of the AMPK complex.

scholarly journals Interleukin-6 signals activating junB and TIS11 gene transcription in a B-cell hybridoma

1991 ◽  
Vol 11 (3) ◽  
pp. 1409-1418
Author(s):  
K Nakajima ◽  
R Wall
Keyword(s):  
Signal Transduction ◽  
Protein Kinase ◽  
B Cell ◽  
Tyrosine Phosphorylation ◽  
Gene Transcription ◽  
Interleukin 6 ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Primary Response ◽  
Protein Kinase Activity

The events in interleukin-6 (IL-6) signal transduction leading to primary response gene activation were analyzed in murine B-cell hybridoma and plasmacytoma cells which require IL-6 for growth. IL-6 stimulation of IL-6-deprived cells resulted in the rapid and transient tyrosine phosphorylation of a 160-kDa cellular protein (p160). This was followed by the highly selective induction of two primary response genes, junB/AP-1 transcription factor and TIS11. junB and TIS11 inductions were unaffected by cycloheximide, suggesting that posttranslational modifications accounted for their activation. Activation of junB and TIS11 transcription required rapid tyrosine kinase activity as well as a different protein kinase activity sensitive to the potent kinase inhibitor, H7, and activated following p160 tyrosine phosphorylation. This H7-sensitive kinase appears to be distinct from any well-characterized protein kinase-second messenger system. On the basis of these findings, we propose that IL-6-induced signal transduction proceeds through a novel protein kinase cascade which activates junB and TIS11 gene transcription.

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