A novel protein kinase CK2 substrate indicates CK2 is not directly stimulated by polyamines in vivo.
The activity of the protein kinase (CK2) is enhanced in vitro by the binding of polyamines to the CK2beta regulatory subunit. The overexpression of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, also elevates CK2 kinase activity in primary keratinocytes and tissues of K6/ODC transgenic mice. In an effort to better characterize the mechanisms by which polyamines may affect CK2 in vivo, we constructed a transfectable CK2 substrate cDNA consisting of the enhanced green fluorescence protein appended with a canonical CK2 phosphorylation sequence (EGFP-S). In contrast to unmodified EGFP, the EGFP-S protein was extensively phosphorylated by CK2, and this phosphorylation was stimulated by the polyamine spermine in a dose-dependent manner. The in vivo phosphorylation of EGFP-S was examined in cell lines which inducibly express either wild-type CK2 holoenzyme or a CK2 holoenzyme which contains activating mutations in the polyamine-binding region of its CK2beta regulatory subunit. Neither the overexpression of ODC in either cell line nor the mutation of the CK2beta subunit conferred an increase in CK2 kinase activity as measured by the in vivo phosphorylation of EGFP-S. Rather, our data indicate that polyamines increase total CK2 kinase activity through increases in steady-state levels of both CK2alpha and CK2beta subunits. The overexpression of ODC resulted in a 3-fold increase in steady-state levels of both exogenous and endogenous CK2 transcripts but did not increase the half-life of wild-type or mutated CK2 protein. These data suggest that the regulation of intracellular CK2 by the polyamines may occur through mechanisms distinct from the direct stimulation of CK2 by polyamines in vitro as previously described.
Lawson
Kathryn
K
Lankenau Institute for Medical Research, 100 Lancaster Avenue, Wynnewood, Pennsylvania 19096, USA.
Larentowicz
Laura
L
Artim
Stephen
S
Hayes
Candace S
CS
Gilmour
Susan K
SK
D047390:Casein Kinase II / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D004440:Ecdysone / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006801:Humans; D009955:Ornithine Decarboxylase / Q000096:biosynthesis; D010766:Phosphorylation; D011073:Polyamines / Q000494:pharmacology*; D021122:Protein Subunits / Q000737:chemistry; D012333:RNA, Messenger / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry
D011073:Polyamines; D021122:Protein Subunits; D012333:RNA, Messenger; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D004440:Ecdysone; D047390:Casein Kinase II; D009955:Ornithine Decarboxylase