The reverse in-gel kinase assay to profile physiological kinase substrates

2007 ◽  
Vol 4 (11) ◽  
pp. 957-962 ◽  
Author(s):  
Xiang Li ◽  
Bin Guan ◽  
Minu K Srivastava ◽  
Achuth Padmanabhan ◽  
Brian S Hampton ◽  
...  
Keyword(s):  
Kinase Assay ◽  
Kinase Substrates

Identification of Plant Kinase Substrates Based on Kinase Assay-Linked Phosphoproteomics

2017 ◽  
pp. 327-335
Author(s):  
Chuan-Chih Hsu ◽  
Justine V. Arrington ◽  
Liang Xue ◽  
W. Andy Tao
Keyword(s):  
Kinase Assay ◽  
Kinase Substrates

In-Gel Kinase Assay as a Method to Identify Kinase Substrates

2002 ◽  
Vol 2002 (153) ◽  
pp. pl15-pl15 ◽  
Author(s):  
M. W. Wooten
Keyword(s):  
Kinase Assay ◽  
Kinase Substrates

scholarly journals Proximity Labeling-assisted Identification of Endogenous Kinase Substrates

2020 ◽  
Author(s):  
Tomoya Niinae ◽  
Koshi Imami ◽  
Naoyuki Sugiyama ◽  
Yasushi Ishihama
Keyword(s):  
Protein Kinase ◽  
Large Scale ◽  
Kinase Assay ◽  
Missense Mutations ◽  
Sequence Motifs ◽  
Single Experiment ◽  
Kinase Substrate ◽  
Endogenous Substrates ◽  
Kinase Substrates

AbstractMass spectrometry-based phosphoproteomics can identify more than 10,000 phosphorylated sites in a single experiment. But, despite the fact that enormous phosphosite information has been accumulated in public repositories, protein kinase-substrate relationships remain largely unknown. Here, we describe a method to identify endogenous substrates of kinases by means of proximity labeling. We used a proximity-dependent biotin identification approach, called BioID, in combination with kinase-perturbed phosphoproteomics profiling and phosphorylation sequence motifs derived from in vitro kinase assay to find molecules that interact with a target kinase, that show altered phosphorylation in response to kinase perturbation, and that are directly phosphorylated by the kinase in vitro; i.e., endogenous kinase substrates. Application of this methodology to casein kinase 2 (CK2) and protein kinase A (PKA) identified 33 and 52 putative substrates, respectively. We also show that known cancer-associated missense mutations near phosphosites of substrates affect phosphorylation by CK2 or PKA, and thus might alter downstream signaling in cancer cells bearing these mutations.This study extends our knowledge of kinase-substrate networks by proposing a new large-scale approach to identify endogenous substrates of kinases.

scholarly journals Sensitive kinase assay linked with phosphoproteomics for identifying direct kinase substrates

2012 ◽  
Vol 109 (15) ◽  
pp. 5615-5620 ◽  
Author(s):  
L. Xue ◽  
W.-H. Wang ◽  
A. Iliuk ◽  
L. Hu ◽  
J. A. Galan ◽  
...  
Keyword(s):  
Kinase Assay ◽  
Kinase Substrates

scholarly journals Identifying Kinase Substrates via a Heavy ATP Kinase Assay and Quantitative Mass Spectrometry

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
André C. Müller ◽  
Roberto Giambruno ◽  
Juliane Weißer ◽  
Peter Májek ◽  
Alexandre Hofer ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Kinase Assay ◽  
Quantitative Mass Spectrometry ◽  
Kinase Substrates

scholarly journals Identification of Direct Tyrosine Kinase Substrates Based on Protein Kinase Assay-Linked Phosphoproteomics

2013 ◽  
Vol 12 (10) ◽  
pp. 2969-2980 ◽  
Author(s):  
Liang Xue ◽  
Robert L. Geahlen ◽  
W. Andy Tao
Keyword(s):  
Protein Kinase ◽  
Tyrosine Kinase ◽  
Kinase Assay ◽  
Kinase Substrates

scholarly journals The U69 Gene of Human Herpesvirus 6 Encodes a Protein Kinase Which Can Confer Ganciclovir Sensitivity to Baculoviruses

1999 ◽  
Vol 73 (4) ◽  
pp. 3284-3291 ◽  
Author(s):  
Azeem Ansari ◽  
Vincent C. Emery
Keyword(s):  
Protein Kinase ◽  
Antiviral Drug ◽  
Human Herpesvirus ◽  
Kinase Assay ◽  
Human Herpesvirus 6 ◽  
Reading Frame ◽  
Kinase Substrate ◽  
Michaelis Constants ◽  
Herpesvirus 6 ◽  
Kinase Substrates

ABSTRACT The protein encoded by the U69 open reading frame (ORF) of human herpesvirus 6 (HHV-6) has been predicted to be a protein kinase. To investigate its functional properties, we have expressed the U69 ORFs from both HHV-6 variants, A and B, by using recombinant baculoviruses (BV6AU69 and BV6BU69). Nickel agarose and antibody affinity chromatography was used to purify the proteins to homogeneity and when incubated with [γ-32P]ATP, both U69 proteins became phosphorylated on predominantly serine residues. These data strongly suggest that U69 is a protein kinase which autophosphorylates. The phosphorylation reaction was optimal at physiological pH and low NaCl concentrations. It required the presence of Mg2+ or Mn2+, and Mg2+ was able to support phosphorylation over a wider range of concentrations than Mn2+. Both ATP and GTP could donate phosphate in the protein kinase assay and the former was more efficient. U69 was capable of phosphorylating histone and casein (serine/threonine kinase substrates) but not enolase (a tyrosine kinase substrate). For the autophosphorylation reaction, the Michaelis constants for ATP of baculovirus-expressed HHV-6A and HHV-6B U69 were calculated to be 44 and 11 μM, respectively. U69 is a homologue of the UL97 gene encoded by human cytomegalovirus which has been shown to phosphorylate the antiviral drug ganciclovir (GCV). We analyzed whether the U69 ORF alone was capable of conferring GCV sensitivity on baculoviruses BV6AU69 and BV6BU69. In plaque reduction experiments, these baculoviruses displayed a GCV-sensitive phenotype compared to a control baculovirus (BVLacZ). The 50% inhibitory concentrations (IC50) of BV6AU69 and BV6BU69 were calculated to be 0.35 and 0.26 mM, respectively, whereas the control baculovirus had an IC50 of >1.4 mM. This shows that the U69 gene product is the only one required to confer GCV sensitivity on baculovirus.

Protein-tyrosine Kinase p72 syk Is Activated by Platelet Activating Factor in Platelets

1994 ◽  
Vol 72 (06) ◽  
pp. 937-941 ◽  
Author(s):  
Karim Rezaul ◽  
Shigeru Yanagi ◽  
Kiyonao Sada ◽  
Takanobu Taniguchi ◽  
Hirohei Yamamura
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Protein Tyrosine Kinase ◽  
Kinase Inhibitor ◽  
Critical Role ◽  
Platelet Activating Factor ◽  
Kinase Assay ◽  
Potential Candidate ◽  
Protein Tyrosine ◽  
Induced Aggregation

SummaryIt has been demonstrated that activation of platelets by platelet-activating factor (PAF) results in a dramatic increase in tyrosine phosphorylation of several cellular proteins. We report here that p72 syk is a potential candidate for the protein-tyrosine phosphorylation following PAF stimulation in porcine platelets. Immunoprecipitation kinase assay revealed that PAF stimulation resulted in a rapid activation of p72 syk which peaked at 10 s. The level of activation was found to be dose dependent and could be completely inhibited by the PAF receptor antagonist, CV3988. Phosphorylation at the tyrosine residues of p72 syk coincided with activation of yllsyk. Pretreatment of platelets with aspirin and apyrase did not affect PAF induced activation of p72 syk .Furthermore, genistein, a potent protein-tyrosine-kinase inhibitor, diminished PAF-induced p72 syk activation and Ca2+ mobilization as well as platelet aggregation. These results suggest that p72 syk may play a critical role in PAF-induced aggregation, possibly through regulation of Ca2+ mobilization.

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