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Ser243 in the activation loop of the Yck2 CK1 protein kinase may be an inhibitory phosphorylation site
Author(s) -
Phillips Joshua,
Larkin Lauren,
Robinson Lucy,
Brame Cynthia
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.566.6
Subject(s) - biology , phosphorylation , mutant , gene , casein kinase 2 , kinase , protein kinase a , casein kinase 1 , fusion protein , mutagenesis , wild type , microbiology and biotechnology , fusion gene , genetics , mitogen activated protein kinase kinase , recombinant dna
The casein kinase 1 (CK1) protein kinase family regulates diverse processes including chromosome segregation, vesicular trafficking, and cell division. Regulatory mechanisms controlling activity of most CK1s are unknown. Using the CK1 Yck2 from budding yeast, we tested the possibility that phosphorylation of serine 243 (S243) in the activation loop can negatively affect activity. S243 was altered to alanine (S243A) and aspartate (S243D) in a GFP:YCK2 fusion via site‐directed mutagenesis. The mutant alleles were expressed in S. cerevisiae to examine function and localization of the encoded variants. From low and high copy vectors, S243A provides normal Yck function and localization. S243D also exhibits normal localization. It supports normal growth from high copy but reduced growth from low copy. In vitro immune complex protein kinase assays using a beta‐galactosidase fusion to Yck2 showed only slight differences between both variant proteins and the wild‐type protein in standard conditions. We integrated the mutant alleles by gene replacement. S243A appears to provide better function than the wild‐type Yck2 gene whereas S243D results in impaired growth. We plan to test in vitro activity of both variants at different temperatures to assess differences that result in altered biological activity and purify Yck2 to directly determine whether S243 is phosphorylated in cells. Supported by NSF grant MCB‐0517204.