scholarly journals Substitution of the Autophosphorylation Site Thr516with a Negatively Charged Residue Confers Constitutive Activity to Mouse 3-Phosphoinositide-dependent Protein Kinase-1 in Cells

2002 ◽  
Vol 277 (19) ◽  
pp. 16632-16638 ◽  
Author(s):  
Michael J. Wick ◽  
KeriLyn R. Wick ◽  
Hui Chen ◽  
Huili He ◽  
Lily Q. Dong ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Constitutive Activity ◽  
Dependent Protein Kinase ◽  
Charged Residue ◽  
Dependent Protein ◽  
Autophosphorylation Site ◽  
In Cells

scholarly journals Inhibition of E2F activity by the cyclin-dependent protein kinase inhibitor p21 in cells expressing or lacking a functional retinoblastoma protein.

1996 ◽  
Vol 16 (6) ◽  
pp. 2987-2997 ◽  
Author(s):  
G P Dimri ◽  
M Nakanishi ◽  
P Y Desprez ◽  
J R Smith ◽  
J Campisi
Keyword(s):  
Protein Kinase ◽  
Kinase Inhibitor ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Wild Type ◽  
Proliferating Cells ◽  
Dependent Protein Kinase ◽  
Nuclear Extracts ◽  
Dependent Protein ◽  
In Cells

p21Sdi1/WAF1/Cip1 inhibits cyclin-dependent protein kinases and cell proliferation. p21 is presumed to inhibit growth by preventing the phosphorylation of growth-regulatory proteins, including the retinoblastoma tumor suppressor protein (pRb). The ultimate effector(s) of p21 growth inhibition, however, is largely a matter of conjecture. We show that p21 inhibits the activity of E2F, an essential growth-stimulatory transcription factor that is negatively regulated by unphosphorylated pRb. p21 suppressed the activity of E2F-responsive promoters (dihydrofolate reductase and cdc2), but E2F-unresponsive promoters (c-fos and simian virus 40 early) were unaffected. Moreover, the simian virus 40 early promoter was rendered p21 suppressible by introducing wild-type, but not mutant, E2F binding sites; p21 deletion mutants showed good agreement in their abilities to inhibit E2F transactivation and DNA synthesis; and E2F-1 (which binds pRb), but not E2F-4 (which does not), reversed both inhibitory effects of p21. Despite the central role for pRb in regulating E2F, p21 suppressed growth and E2F activity in cells lacking a functional pRb. Moreover, p21 protein (wild type but not mutant) specifically disrupted an E2F-cyclin-dependent protein kinase 2-p107 DNA binding complex in nuclear extracts of proliferating cells, whether or not they expressed normal pRb. Thus, E2F is a critical target and ultimate effector of p21 action, and pRb is not essential for the inhibition of growth or E2F-dependent transcription.

scholarly journals Upregulation of STAT1 protein in cells lacking or expressing mutants of the double-stranded RNA-dependent protein kinase PKR

1999 ◽  
Vol 262 (1) ◽  
pp. 149-154 ◽  
Author(s):  
Nancy Wai Ning Tam ◽  
Tetsu Ishii ◽  
Suiyang Li ◽  
Andrew Hoi-Tao Wong ◽  
Andrew R. Cuddihy ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Dependent Protein Kinase ◽  
Double Stranded Rna ◽  
Dependent Protein ◽  
Stat1 Protein ◽  
In Cells

scholarly journals Interleukin-1 beta-converting enzyme-like protease cleaves DNA-dependent protein kinase in cytotoxic T cell killing.

1996 ◽  
Vol 184 (2) ◽  
pp. 619-626 ◽  
Author(s):  
Q Song ◽  
S R Burrows ◽  
G Smith ◽  
S P Lees-Miller ◽  
S Kumar ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Defense Mechanism ◽  
Intact Cell ◽  
Granzyme B ◽  
Interleukin 1 ◽  
Cytotoxic T Cell ◽  
Converting Enzyme ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
In Cells

Cytotoxic T cells (CTL) represent the major defense mechanism against the spread of virus infection. It is believed that the pore-forming protein, perforin, facilitates the entry of a series of serine proteases (particularly granzyme B) into the target cell which ultimately leads to DNA fragmentation and apoptosis. We demonstrate here that during CTL-mediated cytolysis the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), an enzyme implicated in the repair of double strand breaks in DNA, is specifically cleaved by an interleukin (IL)-1 beta-converting enzyme (ICE)-like protease. A serine protease inhibitor, 3,4-dichloroisocoumarin (DCl), which is known to block granzyme B activity, inhibited CTL-induced apoptosis and prevented the degradation of DNA-PKcs in cells but failed to prevent the degradation of purified DNA-PKcs by CTL extracts. However, Tyr-Val-Ala-Asp-CH2Cl (YVAD-CMK) and other cysteine protease inhibitors prevented the degradation of purified DNA-PKcs by CTL extracts. Furthermore, incubation of DNA-PKcs with granzyme B did not produce the same cleavage pattern observed in cells undergoing apoptosis and when this substrate was incubated with either CTL extracts or the ICE-like protease, CPP32. Sequence analysis revealed that the cleavage site in DNA-PKcs during CTL killing was the same as that when this substrate was exposed to CPP32. This study demonstrates for the first time that the cleavage of DNA-PKcs in this intact cell system is exclusively due to an ICE-like protease.

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 Calcium-stimulated Autophosphorylation Site of Plant Chimeric Calcium/Calmodulin-dependent Protein Kinase

2001 ◽  
Vol 276 (35) ◽  
pp. 32940-32947 ◽  
Author(s):  
P. V. Sathyanarayanan ◽  
William F. Siems ◽  
Jeffrey P. Jones ◽  
B. W. Poovaiah
Keyword(s):  
Protein Kinase ◽  
Dependent Protein Kinase ◽  
Calcium Calmodulin Dependent ◽  
Dependent Protein ◽  
Autophosphorylation Site
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