scholarly journals Identification of -R-X-(X)-S/T-X 3-S/T as consensus sequence motif for autophosphorylation-dependent protein kinase.

1995 ◽  
Vol 270 (7) ◽  
pp. 3462
Author(s):  
Shiaw-Der Yang ◽  
Tze-Jen Huang
Keyword(s):  
Protein Kinase ◽  
Consensus Sequence ◽  
Sequence Motif ◽  
Dependent Protein Kinase ◽  
Dependent Protein

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 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.

cAMP-dependent protein kinase defines a family of enzymes

1993 ◽  
Vol 340 (1293) ◽  
pp. 315-324 ◽  
Keyword(s):  
Protein Kinase ◽  
Consensus Sequence ◽  
Protein Kinase Inhibitors ◽  
Catalytic Subunit ◽  
Affinity Binding ◽  
Dependent Protein Kinase ◽  
High Affinity Binding ◽  
High Affinity ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein

The structure of the recombinant mouse catalytic subunit of cAMP-dependent protein kinase is reviewed with particular emphasis on the overall features and specific amino acids that are shared by all members of the eukaryotic protein kinase family. The crystal structure of a ternary complex containing both MgATP and a twenty-residue inhibitor peptide defines the precise role of the conserved residues that are clustered at the active site. In addition to catalysing the post-translational modification of other proteins, the catalytic subunit is itself subject to covalent modifications. It is a phosphoprotein and is also myristylated at its amino terminus. The enzyme when crystallized in the presence of detergent shows a detergent molecule bound to an acyl pocket that is presumably occupied by the myristyl moiety in the mammalian enzyme. When expressed in E.coli , the catalytic subunit is autophosphorylated at four sites. Two stable phosphates at Ser338 and Thrl97 interact with multiple protein side chains thus explaining why they are inaccessible to phosphatases. Although all substrates and inhibitors of the catalytic subunit share a general minimum consensus sequence, the high affinity binding of protein inhibitors such as the regulatory subunits and the heat stable protein kinase inhibitors require additional determinants that lie beyond the consensus site. These two physiological inhibitors of the catalytic subunit appear to use different sites to achieve high-affinity binding.

scholarly journals Transcription factor Hes1 modulates osteoarthritis development in cooperation with calcium/calmodulin-dependent protein kinase 2

2015 ◽  
Vol 112 (10) ◽  
pp. 3080-3085 ◽  
Author(s):  
Shurei Sugita ◽  
Yoko Hosaka ◽  
Keita Okada ◽  
Daisuke Mori ◽  
Fumiko Yano ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Protein Kinase ◽  
Notch Signaling ◽  
Target Genes ◽  
Consensus Sequence ◽  
Type Ii Collagen ◽  
Dependent Protein Kinase ◽  
Calcium Calmodulin Dependent ◽  
Chondroprogenitor Cells ◽  
Dependent Protein

Notch signaling modulates skeletal formation and pathogenesis of osteoarthritis (OA) through induction of catabolic factors. Here we examined roles of Hes1, a transcription factor and important target of Notch signaling, in these processes. SRY-box containing gene 9 (Sox9)-Cre mice were mated with Hes1fl/fl mice to generate tissue-specific deletion of Hes1 from chondroprogenitor cells; this deletion caused no obvious abnormality in the perinatal period. Notably, OA development was suppressed when Hes1 was deleted from articular cartilage after skeletal growth in type II collagen (Col2a1)-CreERT;Hes1fl/fl mice. In cultured chondrocytes, Hes1 induced metallopeptidase with thrombospondin type 1 motif, 5 (Adamts5) and matrix metalloproteinase-13 (Mmp13), which are catabolic enzymes that break down cartilage matrix. ChIP-seq and luciferase assays identified Hes1-responsive regions in intronic sites of both genes; the region in the ADAMTS5 gene contained a typical consensus sequence for Hes1 binding, whereas that in the MMP13 gene did not. Additionally, microarray analysis, together with the ChIP-seq, revealed novel Hes1 target genes, including Il6 and Il1rl1, coding a receptor for IL-33. We further identified calcium/calmodulin-dependent protein kinase 2δ (CaMK2δ) as a cofactor of Hes1; CaMK2δ was activated during OA development, formed a protein complex with Hes1, and switched it from a transcriptional repressor to a transcriptional activator to induce cartilage catabolic factors. Therefore, Hes1 cooperated with CaMK2δ to modulate OA pathogenesis through induction of catabolic factors, including Adamts5, Mmp13, Il6, and Il1rl1. Our findings have contributed to further understanding of the molecular pathophysiology of OA, and may provide the basis for development of novel treatments for joint disorders.

Regulation of Kv4.3 currents by Ca2+/calmodulin-dependent protein kinase II

2005 ◽  
Vol 288 (2) ◽  
pp. C304-C313 ◽  
Author(s):  
Gerard P. Sergeant ◽  
Susumu Ohya ◽  
James A. Reihill ◽  
Brian A. Perrino ◽  
Gregory C. Amberg ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Consensus Sequence ◽  
Terminal Region ◽  
Transient Outward Current ◽  
Inhibitory Peptide ◽  
Dependent Protein Kinase ◽  
Recovery From Inactivation ◽  
Protein Kinase Ii ◽  
Dependent Protein ◽  
Rate Of Recovery

The voltage-dependent K+ channel 4.3 (Kv4.3) is one of the major molecular correlates encoding a class of rapidly inactivating K+ currents, including the transient outward current in the heart ( Ito) and A currents ( IA) in neuronal and smooth muscle preparations. Recent studies have shown that Ito in human atrial myocytes and IA in murine colonic myocytes are modulated by Ca2+/calmodulin-dependent protein kinase II (CaMKII); however, the molecular target of CaMKII in these studies has not been elucidated. We performed experiments to investigate whether CaMKII could regulate Kv4.3 currents directly. Inclusion of the autothiophosphorylated form of CaMKII in the patch pipette (10 nM) prolonged Kv4.3 currents such that the time required to reach 50% inactivation from peak more than doubled, with positive shifts in voltage dependence of both activation and inactivation. In contrast, the rate of recovery from inactivation was accelerated under these conditions. CaMKII-inhibitory peptide or KN-93 produced effects opposite to that above; thus the rate of inactivation was increased, and recovery from inactivation decreased. A number of mutagenesis experiments were conducted on the three candidate CaMKII consensus sequence sites on the channel. Mutations at S550A, located at the COOH-terminal region of the channel, resulted in currents that inactivated more rapidly but recovered from inactivation at a slower rate than that of wild-type controls. In addition, these currents were unaffected by dialysis with either autothiophosphorylated CaMKII or the specific inhibitory peptide of CaMKII, suggesting that CaMKII slows the inactivation and accelerates the rate of recovery from inactivation of Kv4.3 currents by a direct effect at S550A, located at the COOH-terminal region of the channel.

Sign in / Sign up

Export Citation Format

Share Document