Identification of a splicing isoform of mammalian Wee1
by Yaping Yu
THESIS
1999
Ph.D. Biochemistry
XV, 161 leaves : ill. (some col.) ; 30 cm
Abstract
In higher eukaryotes, cell cycle transitions are promoted by cyclin dependent kinases (Cdks) of which are a series of protein Ser/Thr kinases and the activities of Cdks are tightly regulated by several complex mechanisms. Entry into mitosis is negatively regulated by the inhibitory phosphorylation of tyrosine 15 on Cdc2 by Wee1 protein kinase. Wee1 protein kinase is widely conserved in various species. During screening of a rat brain cDNA library, a rat Wee1 isoform was identified. Sequence analysis indicated that the isoform is due to the alternative splicing. The rat Wee1 isoform was successfully amplified by polymerase chain reaction and reverse transcriptase polymerase chain reaction in all tested rat tissues and cell lines. In Northern blot analysis using a rat Wee1 cDNA probe, two...[ Read more ]
In higher eukaryotes, cell cycle transitions are promoted by cyclin dependent kinases (Cdks) of which are a series of protein Ser/Thr kinases and the activities of Cdks are tightly regulated by several complex mechanisms. Entry into mitosis is negatively regulated by the inhibitory phosphorylation of tyrosine 15 on Cdc2 by Wee1 protein kinase. Wee1 protein kinase is widely conserved in various species. During screening of a rat brain cDNA library, a rat Wee1 isoform was identified. Sequence analysis indicated that the isoform is due to the alternative splicing. The rat Wee1 isoform was successfully amplified by polymerase chain reaction and reverse transcriptase polymerase chain reaction in all tested rat tissues and cell lines. In Northern blot analysis using a rat Wee1 cDNA probe, two mRNA transcripts were detected in various rat tissues and cells, representing Wee1 and Wee1 isoform respectively. Both rat Wee 1 (RWee1) and rat Wee1 isoform (RWeeli) were isolated during the library screening from a rat brain cDNA library and expressed in vitro. The expression of RWee1 and RWeeli was confirmed by Western blot analysis. Bacterially expressed RWeel and RWee1i can also phosphorylate Cdc2 peptide substrate at tyrosine 15 with similar rates. Both endogenous proteins of the two forms of Wee1 were purified and separated from rat thymus by column chromatography, and they were found to phosphorylate Cdk2 protein in vitro.
The functional effects of RWee1 and RWeeli on the phosphorylation of Cdk5 and Cdk2 kinases were examined. At a concentration of RWeel or RWeeli sufficient to maximally inactivate reconstituted Cdk2 kinase, little changes of the kinase activity of reconstituted Cdk5 were observed. The level of Cdk5 phosphorylation by Wee1 is much lower than Cdk2. In addition, the reconstituted active Cdk5/p25 complex is more refractory to Wee1 phosphorylation compared to monomeric Cdk5. However, RWeel and RWeeli exhibited higher efficiency of phosphorylation and inhibition of baculovirus co-expressed Cdk5/p35 complex and this inhibition was demonstrated to be due to phosphorylation of Cdk5. Thus, it warrants serious consideration that Cdk5 can be converted into an efficient Wee1 kinase substrate by association with certain protein factors.
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