Document Detail


Pseudosubstrate sequence may not be critical for autoinhibition of smooth muscle myosin light chain kinase.
MedLine Citation:
PMID:  7796810     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
It has been hypothesized that basic residues in the autoinhibitory region of myosin light chain (MLC) kinase, which resemble the substrate sequence, interact with the catalytic core via charge interaction and thus inhibit the kinase activity (pseudosubstrate inhibitory hypothesis). In the present study, we produced seven MLC kinase mutants in which the residues in the autoinhibitory region are deleted to various extents, and determined the residues crucial for the autoinhibition of the kinase activity. The activities of MT799 (1-799) and MT796 (1-796) were completely inhibited, whereas MT793 (1-793), MT791 (1-791), MT787 (1-787) and MT783 (1-783) were constitutively active. The tryptic proteolysis of MT799 and MT796 activated the kinase activity, presumably due to the removal of the residues essential for autoinhibition. The mutants which showed the constitutively active kinase activity were not further activated by tryptic proteolysis, suggesting that the residues crucial for autoinhibition were already deleted. On the other hand, MT795 (1-795) was partially constitutively active (33% of maximum activity) and the tryptic proteolysis further activated the enzyme activity, suggesting that MT795 loses part of the residues essential for autoinhibition. The substitution of the residues Tyr794-Met795 but not Lys793 of untruncated MLC kinase significantly increased the Ca2+/calmodulin-independent kinase activity. These results clearly show that the region Tyr794-Met795-Ala796 is critical for autoinhibition. This study shows that the pseudosubstrate sequence is not critical for the autoinhibition mechanism of MLC kinase.
Authors:
M Tanaka; R Ikebe; M Matsuura; M Ikebe
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  The EMBO journal     Volume:  14     ISSN:  0261-4189     ISO Abbreviation:  EMBO J.     Publication Date:  1995 Jun 
Date Detail:
Created Date:  1995-08-03     Completed Date:  1995-08-03     Revised Date:  2009-11-19    
Medline Journal Info:
Nlm Unique ID:  8208664     Medline TA:  EMBO J     Country:  ENGLAND    
Other Details:
Languages:  eng     Pagination:  2839-46     Citation Subset:  IM    
Affiliation:
Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Sequence
Animals
Base Sequence
Calcium Chloride / metabolism
Calmodulin / metabolism
Molecular Sequence Data
Molecular Weight
Muscle, Smooth / enzymology*
Myosin-Light-Chain Kinase / chemistry,  genetics,  isolation & purification,  metabolism*
Recombinant Proteins / biosynthesis,  isolation & purification
Sequence Deletion / physiology
Substrate Specificity
Trypsin
Turkeys
Grant Support
ID/Acronym/Agency:
HL 37117/HL/NHLBI NIH HHS; HL 47530/HL/NHLBI NIH HHS
Chemical
Reg. No./Substance:
0/Calmodulin; 0/Recombinant Proteins; 10043-52-4/Calcium Chloride; EC 2.7.11.18/Myosin-Light-Chain Kinase; EC 3.4.21.4/Trypsin
Comments/Corrections

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