Probing the Binding of Indolactam-V to Protein Kinase C through Site-Directed Mutagenesis and Computational Docking Simulations

1999 ◽  
Vol 42 (18) ◽  
pp. 3436-3446 ◽  
Author(s):  
Shaomeng Wang ◽  
Ming Liu ◽  
Nancy E. Lewin ◽  
Patricia S. Lorenzo ◽  
Dipak Bhattacharrya ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Computational Docking ◽  
Docking Simulations ◽  
Kinase C

scholarly journals Residues in the Second Cysteine-rich Region of Protein Kinase C δ Relevant to Phorbol Ester Binding as Revealed by Site-directed Mutagenesis

1995 ◽  
Vol 270 (37) ◽  
pp. 21852-21859 ◽  
Author(s):  
Marcelo G. Kazanietz ◽  
Shaomeng Wang ◽  
George W. A. Milne ◽  
Nancy E. Lewin ◽  
Howard L. Liu ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phorbol Ester ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Rich Region ◽  
Kinase C

Molecular Modeling and Site-Directed Mutagenesis Studies of a Phorbol Ester-Binding Site in Protein Kinase C

1996 ◽  
Vol 39 (13) ◽  
pp. 2541-2553 ◽  
Author(s):  
Shaomeng Wang ◽  
Marcelo G. Kazanietz ◽  
Peter M. Blumberg ◽  
Victor E. Marquez ◽  
G. W. A. Milne
Keyword(s):  
Protein Kinase C ◽  
Molecular Modeling ◽  
Protein Kinase ◽  
Binding Site ◽  
Phorbol Ester ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Kinase C

scholarly journals Analysis of Gz alpha by site-directed mutagenesis. Sites and specificity of protein kinase C-dependent phosphorylation.

1993 ◽  
Vol 268 (5) ◽  
pp. 3494-3498
Author(s):  
K.M. Lounsbury ◽  
B. Schlegel ◽  
M. Poncz ◽  
L.F. Brass ◽  
D.R. Manning
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Kinase C

scholarly journals Mechanisms of Phorbol Ester Toxicity, Determined by Molecular Modelling

2020 ◽  
pp. 10-24
Author(s):  
A. Wakandigara ◽  
L. R. M. Nhamo ◽  
J. Kugara ◽  
P. Mushonga
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phorbol Ester ◽  
Phorbol Esters ◽  
Computer Modelling ◽  
Free Acid ◽  
Cell Damage ◽  
Hydrophobic Surface ◽  
Computational Docking ◽  
Kinase C

Phorbol esters are toxic phytochemicals, whose main biological target is protein kinase C. They bind irreversibly to the protein, causing cell damage. Using computer modelling, we have determined, for the first time, features and mechanisms that lead to the toxicity of phorbol esters. Protein kinase C – delta (PKC-δ) was used as a target protein in computational docking studies with phorbol esters that differ in molecular structure. Binding conformations and stability of ester linkages were analyzed to evaluate their relationship with experimental observations. Results show that an active phorbol ester must exhibit two features: interaction with specific amino acid residues at the binding site and covering the area with a hydrophobic surface. Toxicity of an active phorbol ester is inversely proportional to the intrinsic reactivity of the ester linkage. Phorbol esters bearing free acid chains can directly activate PKC-δ but jatropha phorbol esters are restricted by their acid-moiety ring formations, suggesting similar mechanism of interaction with other phorbol-ester protein targets.

scholarly journals Mutagenesis analysis of human SM22: characterization of actin binding

2000 ◽  
Vol 89 (5) ◽  
pp. 1985-1990 ◽  
Author(s):  
Yiping Fu ◽  
Hong Wei Liu ◽  
Sean M. Forsythe ◽  
Paul Kogut ◽  
John F. McConville ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Full Length ◽  
Serine Phosphorylation ◽  
Site Directed Mutagenesis ◽  
Actin Binding ◽  
Internal Deletion ◽  
Terminal Domain ◽  
Kinase C

SM22 is a 201-amino acid actin-binding protein expressed at high levels in smooth muscle cells. It has structural homology to calponin, but how SM22 binds to actin remains unknown. We performed site-directed mutagenesis to generate a series of NH2-terminal histidine (His)-tagged mutants of human SM22 in Escherichia coli and used these to analyze the functional importance of potential actin binding domains. Purified full-length recombinant SM22 bound to actin in vitro, as demonstrated by cosedimentation assay. Binding did not vary with calcium concentration. The COOH-terminal domain of SM22 is required for actin affinity, because COOH terminally truncated mutants [SM22-(1–186) and SM22-(1–166)] exhibited markedly reduced cosedimentation with actin, and no actin binding of SM22-(1–151) could be detected. Internal deletion of a putative actin binding site (154-KKAQEHKR-161) partially prevented actin binding, as did point mutation to neutralize either or both pairs of positively charged residues at the ends of this region (KK154LL and/or KR160LL). Internal deletion of amino acids 170–180 or 170–186 also partially or almost completely inhibited actin cosedimentation, respectively. Of the three consensus protein kinase C or casein kinase II phosphorylation sites in SM22, only Ser-181 was readily phosphorylated by protein kinase C in vitro, and such phosphorylation greatly decreased actin binding. Substitution of Ser-181 to aspartic acid (to mimic serine phosphorylation) also reduced actin binding. Immunostains of transiently transfected airway myocytes revealed that full-length NH2-terminal FLAG-tagged SM22 colocalizes with actin filaments, whereas FLAG-SM22-(1–151) does not. These data confirm that SM22 binds to actin in vitro and in vivo and, for the first time, demonstrate that multiple regions within the COOH-terminal domain are required for full actin affinity.

scholarly journals The Phosphoprotein of Rabies Virus Is Phosphorylated by a Unique Cellular Protein Kinase and Specific Isomers of Protein Kinase C

2000 ◽  
Vol 74 (1) ◽  
pp. 91-98 ◽  
Author(s):  
Ashim K. Gupta ◽  
Danielle Blondel ◽  
Suresh Choudhary ◽  
Amiya K. Banerjee
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Protein Kinases ◽  
Rabies Virus ◽  
Cell Extract ◽  
Site Directed Mutagenesis ◽  
Virus Protein ◽  
P Gene ◽  
Kinase C ◽  
P Protein

ABSTRACT The phosphoprotein (P) gene of rabies virus (CVS strain) was cloned and expressed in bacteria. The purified protein was used as the substrate for phosphorylation by the protein kinase(s) present in cell extract prepared from rat brain. Two distinct types of protein kinases, staurosporin sensitive and heparin sensitive, were found to phosphorylate the P protein in vitro by the cell extract. Interestingly, the heparin-sensitive kinase was not the ubiquitous casein kinase II present in a variety of cell types. Further purification of the cell fractions revealed that the protein kinase C (PKC) isomers constitute the staurosporin-sensitive kinases α, β, γ, and ζ, with the PKCγ isomer being the most effective in phosphorylating the P protein. A unique heparin-sensitive kinase was characterized as a 71-kDa protein with biochemical properties not demonstrated by any known protein kinases stored in the protein data bank. This protein kinase, designated RVPK (rabies virus protein kinase), phosphorylates P protein (36 kDa) and alters its mobility in gel to migrate at 40 kDa. In contrast, the PKC isoforms do not change the mobility of unphosphorylated P protein. RVPK appears to be packaged in the purified virions, to display biochemical characteristics similar to those of the cell-purified RVPK, and to similarly alter the mobility of endogenous P protein upon phosphorylation. By site-directed mutagenesis, the sites of phosphorylation of RVPK were mapped at S63 and S64, whereas PKC isomers phosphorylated at S162, S210, and S271. Involvement of a unique protein kinase in phosphorylating rabies virus P protein indicates its important role in the structure and function of the protein and consequently in the life cycle of the virus.

scholarly journals Annexin V inhibits protein kinase C activity via a mechanism of phospholipid sequestration

1998 ◽  
Vol 330 (3) ◽  
pp. 1277-1282 ◽  
Author(s):  
Thierry DUBOIS ◽  
Jean-Paul MIRA ◽  
Denis FELIERS ◽  
Egle SOLITO ◽  
Françoise RUSSO-MARIE ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Binding Site ◽  
Annexin V ◽  
Jurkat Cells ◽  
Site Directed Mutagenesis ◽  
Kinase C ◽  
Protein Kinase C Activity ◽  
Mechanism Of Inhibition

In this study, we assessed the role of annexin V, a Ca2+-dependent phospholipid-binding protein, as a regulator of protein kinase C (PKC) and characterized its mechanism of inhibition. Several mutants obtained by oligonucleotide site-directed mutagenesis were tested in vitro on PKC activity in cytosolic fractions from Jurkat cells and on purified PKCα. Annexin V inhibited phosphorylation of annexin II by endogenous PKC and phosphorylation of myelin basic protein by PKCα. In both systems, the use of single Ca2+-binding-site mutants of annexin V led to a partial reversal of inhibition, and the Ca2+-binding site located in the first domain of annexin V was found to have the most important role. An increase in the number of mutated Ca2+-binding sites led to a greater loss of inhibition. These results corroborated those showing the progressive loss of binding of these mutants to phospholipid liposomes. In conclusion, we show that PKC inhibition by annexin V is the consequence of a mechanism involving phospholipid sequestration by annexin V, and that the Ca2+-binding site located in domain 1 of annexin V plays a predominant role in this process. In addition, we show that the R122AIK site, which may act analogously to a PKC-inhibitory pseudosubstrate site, is not involved in PKC inhibition, and that a peptide corresponding to the C-terminal tail of annexin V inhibits PKC activity but to a lesser extent than annexin V itself.

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