scholarly journals Identification of the regulatory autophosphorylation site of autophosphorylation-dependent protein kinase (auto-kinase)

1998 ◽  
Vol 334 (1) ◽  
pp. 121-131 ◽  
Author(s):  
Jau-Song YU ◽  
Wei-Jen CHEN ◽  
Mei-Hui NI ◽  
Wen-Hsiung CHAN ◽  
Shiaw-Der YANG
Keyword(s):  
Protein Kinase ◽  
Specific Antibody ◽  
Consensus Sequence ◽  
Sequence Motif ◽  
Activation Process ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Catalytic Fragment ◽  
Substrate Consensus Sequence ◽  
Autophosphorylation Site

Autophosphorylation-dependent protein kinase (auto-kinase) was identified from pig brain and liver on the basis of its unique autophosphorylation/activation property [Yang, Fong, Yu and Liu (1987) J. Biol. Chem. 262, 7034–7040; Yang, Chang and Soderling (1987) J. Biol. Chem. 262, 9421–9427]. Its substrate consensus sequence motif was determined as being -R-X-(X)-S*/T*-X3-S/T-. To characterize auto-kinase further, we partly sequenced the kinase purified from pig liver. The N-terminal sequence (VDGGAKTSDKQKKKAXMTDE) and two internal peptide sequences (EKLRTIV and LQNPEK/ILTP/FI) of auto-kinase were obtained. These sequences identify auto-kinase as a C-terminal catalytic fragment of p21-activated protein kinase 2 (PAK2 or γ-PAK) lacking its N-terminal regulatory region. Auto-kinase can be recognized by an antibody raised against the C-terminal peptide of human PAK2 by immunoblotting. Furthermore the autophosphorylation site sequence of auto-kinase was successfully predicted on the basis of its substrate consensus sequence motif and the known PAK2 sequence, and was further demonstrated to be RST(P)MVGTPYWMAPEVVTR by phosphoamino acid analysis, manual Edman degradation and phosphopeptide mapping via the help of phosphorylation site analysis of a synthetic peptide corresponding to the sequence of PAK2 from residues 396 to 418. During the activation process, auto-kinase autophosphorylates mainly on a single threonine residue Thr402 (according to the sequence numbering of human PAK2). In addition, a phospho-specific antibody against a synthetic phosphopeptide containing this identified sequence was generated and shown to be able to differentially recognize the activated auto-kinase autophosphorylated at Thr402 but not the non-phosphorylated/inactive auto-kinase. Immunoblot analysis with this phospho-specific antibody further revealed that the change in phosphorylation level of Thr402 of auto-kinase was well correlated with the activity change of the kinase during both autophosphorylation/activation and protein phosphatase-mediated dephosphorylation/inactivation processes. Taken together, our results identify Thr402 as the regulatory autophosphorylation site of auto-kinase, which is a C-terminal catalytic fragment of PAK2.

scholarly journals Cyclic AMP-dependent protein kinase inhibits the activity of myogenic helix-loop-helix proteins.

1992 ◽  
Vol 12 (10) ◽  
pp. 4478-4485 ◽  
Author(s):  
L Li ◽  
R Heller-Harrison ◽  
M Czech ◽  
E N Olson
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Transcriptional Activation ◽  
Consensus Sequence ◽  
Signal Transduction Pathway ◽  
Specific Gene ◽  
Dependent Protein Kinase ◽  
Dependent Protein

Differentiation of skeletal muscle cells is inhibited by the cyclic AMP (cAMP) signal transduction pathway. Here we report that the catalytic subunit of cAMP-dependent protein kinase (PKA) can substitute for cAMP and suppress muscle-specific transcription by silencing the activity of the MyoD family of regulatory factors, which includes MyoD, myogenin, myf5, and MRF4. Repression by the PKA catalytic (C) subunit is directed at the consensus sequence CANNTG, the target for DNA binding and transcriptional activation by these myogenic regulators. Phosphopeptide mapping of myogenin in vitro and in vivo revealed two PKA phosphorylation sites, both within the basic region. However, repression of myogenin function by PKA does not require direct phosphorylation of these sites but instead involves an indirect mechanism with one or more intermediate steps. Regulation of the transcriptional activity of the MyoD family by modulation of the cAMP signaling pathway may account for the inhibitory effects of certain peptide growth factors on muscle-specific gene expression and may also determine the responsiveness of different cell types to myogenic conversion by these myogenic regulators.

Cyclic AMP-dependent protein kinase inhibits the activity of myogenic helix-loop-helix proteins

1992 ◽  
Vol 12 (10) ◽  
pp. 4478-4485
Author(s):  
L Li ◽  
R Heller-Harrison ◽  
M Czech ◽  
E N Olson
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Transcriptional Activation ◽  
Consensus Sequence ◽  
Signal Transduction Pathway ◽  
Specific Gene ◽  
Dependent Protein Kinase ◽  
Dependent Protein

Differentiation of skeletal muscle cells is inhibited by the cyclic AMP (cAMP) signal transduction pathway. Here we report that the catalytic subunit of cAMP-dependent protein kinase (PKA) can substitute for cAMP and suppress muscle-specific transcription by silencing the activity of the MyoD family of regulatory factors, which includes MyoD, myogenin, myf5, and MRF4. Repression by the PKA catalytic (C) subunit is directed at the consensus sequence CANNTG, the target for DNA binding and transcriptional activation by these myogenic regulators. Phosphopeptide mapping of myogenin in vitro and in vivo revealed two PKA phosphorylation sites, both within the basic region. However, repression of myogenin function by PKA does not require direct phosphorylation of these sites but instead involves an indirect mechanism with one or more intermediate steps. Regulation of the transcriptional activity of the MyoD family by modulation of the cAMP signaling pathway may account for the inhibitory effects of certain peptide growth factors on muscle-specific gene expression and may also determine the responsiveness of different cell types to myogenic conversion by these myogenic regulators.

Regulation of an epithelial chloride channel by direct phosphorylation and dephosphorylation

1992 ◽  
Vol 263 (1) ◽  
pp. C172-C175 ◽  
Author(s):  
A. L. Finn ◽  
M. L. Gaido ◽  
M. Dillard ◽  
D. L. Brautigan
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Lipid Bilayers ◽  
Okadaic Acid ◽  
Specific Antibody ◽  
Chloride Channel ◽  
Protein Phosphatase ◽  
Dependent Protein Kinase ◽  
Phosphatase 2A ◽  
Dependent Protein

A native chloride channel in Necturus gallbladder epithelial cells is opened by a theophylline-induced rise in cellular cyclic AMP and is closed by removal of theophylline or by addition of specific antibody; however, it does not close if okadaic acid, an inhibitor of protein phosphatases 1 and 2A, is added. The purified channel reconstituted into lipid bilayers closes upon the addition of protein phosphatase 2A and is reopened by the addition of Mg-ATP and the catalytic subunit of cyclic AMP-dependent protein kinase. These results indicate that the channel protein is purified in a phosphorylated state and that its functional characteristics are at least partly controlled by direct phosphorylation and dephosphorylation.

Regulation of Ca2+/calmodulin-dependent protein kinase II catalysis by N-methyl-D-aspartate receptor subunit 2B

2009 ◽  
Vol 419 (1) ◽  
pp. 123-136 ◽  
Author(s):  
Kurup K. Pradeep ◽  
John Cheriyan ◽  
Sudarsana Devi Suma Priya ◽  
Raveendran Rajeevkumar ◽  
Madhavan Mayadevi ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Kinetic Parameters ◽  
Glutathione Transferase ◽  
Long Term Potentiation ◽  
Binding Motif ◽  
Sequence Motif ◽  
Maximal Velocity ◽  
Dependent Protein Kinase ◽  
Protein Kinase Ii ◽  
Dependent Protein

Binding of CaMKII (Ca2+/calmodulin-dependent protein kinase II) to the NR2B subunit of the NMDAR (N-methyl-D-aspartate-type glutamate receptor) in the PSD (postsynaptic density) is essential for the induction of long-term potentiation. In this study, we show that binding of NR2B to the T-site (Thr286-autophosphorylation site binding pocket) of CaMKII regulates its catalysis as reflected in the kinetic parameters. The apparent S0.5 (substrate concentration at half maximal velocity) and Vmax values for ATP were lower for phosphorylation of a GST (glutathione transferase)-fusion of NR2B(1271-1311) (with the phosphorylation site Ser1303) when compared with phosphorylation of the analogous sequence motif from NR2A. The co-operative behaviour exhibited by the CaMKII holoenzyme towards ATP for phosphorylation of GST–NR2A was significantly altered by the interaction with GST–NR2B. Disrupting the T-site-mediated binding by mutagenesis of either NR2B or CaMKII abolished the modulation of CaMKII activity by NR2B. The active site residue of α-CaMKII, Glu96, participates in effecting the modulation. The CaMKII-binding motif of the Drosophila voltage-gated potassium channel Eag interacted with the T-site of CaMKII with lower affinity and caused catalytic modulation to a lesser extent. The kinetic parameters of ATP for the Thr286-autophosphorylation reaction of CaMKII were also altered by NR2B in a similar manner. Interestingly, the NR2B sequence motif caused increased sensitivity of CaMKII activity to ATP, and saturation by lower concentrations of ATP, which, in effect, resulted in a constant level of activity of CaMKII over a broad range of ATP concentrations. Our findings indicate that CaMKII at the PSD may be regulated by bound NR2B in a manner that supports synaptic memories.

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