Document Detail

Phosphorylation of Srp1p, the yeast nuclear localization signal receptor, in vitro and in vivo.
MedLine Citation:
PMID:  9258433     Owner:  NLM     Status:  MEDLINE    
Srp1p, the protein encoded by SRP1 of the yeast Saccharomyces cerevisiae, is a yeast nuclear localization signal (NLS) receptor protein. We have previously reported isolation of a protein kinase from yeast extracts that phosphorylates Srp1p complexed with NLS peptides/proteins. From partial amino acid sequences of the four subunits of the purified kinase, we have now identified this protein kinase to be identical to yeast casein kinase II (CKII). It was previously thought that autophosphorylation of the 36 kDa subunit of the yeast enzyme was stimulated by the substrate, GST-Srp1p. However, with the use of a more refined system, no stimulation of autophosphorylation of the 36 kDa subunit of yeast CKII was observed. Biochemical and mutational analyses localized the in vitro phosphorylation site of Srp1p by CKII to serine 67. It was shown that, in the absence of NLS peptides/proteins, phosphorylation of the intact Srp1p protein is very weak, but deletion of the C-terminal end causes great stimulation of phosphorylation without NLS peptides/proteins. Thus, the CKII phosphorylation site is apparently masked in the intact protein structure by the presence of a C-terminal region, probably between amino acids 403 and 516. Binding of NLS peptides/proteins most likely causes a change in protein conformation, exposing the CKII phosphorylation site. Mutational alterations of serine 67, the CKII phosphorylation site, to valine (S67V) and aspartic acid (S67D) were not found to cause any significant deleterious effects on cell growth. Analysis of in vivo phosphorylation showed that at least 30% of the wild type Srp1p molecules are phosphorylated in growing cells, and that the phosphorylation is mostly at the serine 67 CKII site. The ability of Srp1p purified from E coli and treated with calf intestinal phosphatase to bind a SV40 T-antigen NLS peptide was compared with that of Srp1p which was almost fully phosphorylated by CKII. No significant difference was observed. It appears that NLS binding does not require any phosphorylation of Srp1p, either by CKII or by some other protein kinase.
Y Azuma; K Takio; M M Tabb; L Vu; M Nomura
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Biochimie     Volume:  79     ISSN:  0300-9084     ISO Abbreviation:  Biochimie     Publication Date:  1997 May 
Date Detail:
Created Date:  1997-10-23     Completed Date:  1997-10-23     Revised Date:  2009-11-19    
Medline Journal Info:
Nlm Unique ID:  1264604     Medline TA:  Biochimie     Country:  FRANCE    
Other Details:
Languages:  eng     Pagination:  247-59     Citation Subset:  IM    
Department of Biological Chemistry, University of California, Irvine 92697-1700, USA.
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MeSH Terms
Amino Acid Sequence
Binding Sites
Casein Kinase II
Fungal Proteins / metabolism*
Heat-Shock Proteins*
Molecular Sequence Data
Nuclear Localization Signals
Nuclear Proteins / metabolism
Protein Conformation
Protein-Serine-Threonine Kinases / metabolism*
Recombination, Genetic
Saccharomyces cerevisiae / metabolism
Saccharomyces cerevisiae Proteins*
Structure-Activity Relationship
Grant Support
R376M35949//PHS HHS
Reg. No./Substance:
0/Fungal Proteins; 0/Heat-Shock Proteins; 0/Nuclear Localization Signals; 0/Nuclear Proteins; 0/Saccharomyces cerevisiae Proteins; 0/TIR1 protein, S cerevisiae; EC Kinase II; EC Kinases

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