A Novel Protein Kinase CK2 Substrate Indicates CK2 Is Not Directly Stimulated by Polyamines in Vivo

Abstract

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.

Characterizing the convergence of protein kinase CK2 and caspase-3 reveals isoform-specific phosphorylation of caspase-3 by CK2α′: implications for pathological roles of CK2 in promoting cancer cell survival

“…Under some circumstances, CK2 holoenzyme-mediated phosphorylation can be stimulated by polyamines in in vitro kinase assays and by over-expression of ODC, the rate limiting enzyme of spermine synthesis, in cells [34-36]. However, in the case of caspase-3, over-expression of ODC1 did not promote its phosphorylation (data not shown).…”

Organization and Regulation of Small Conductance Ca2+-activated K+Channel Multiprotein Complexes

“…Although this is the most parsimonious explanation for the results, it is also possible that the K121A mutation may otherwise affect CK2 binding. Polybasic compounds stimulate CK2 activity likely through interactions with the acid cluster on the ␤ subunit (Niefind et al, 2001) (but see Lawson et al, 2006). The presence of positive charges may result in charge shielding of the acid loop that normally inhibits CK2 activity (Niefind et al, 2001).…”

Cellular regulators of protein kinase CK2

“…Some indication has been presented that the stimulation of CK2 activity by polyamines is attributable to an alteration of the quaternary structure of CK2 corresponding to a catalytically active conformation (Valero et al 1995). Recent elegant experiments by Lawson et al (2006) have revealed that polyamines increase total CK2 kinase activity through an increase in the steady levels of both CK2α and CK2β. Thus, these data suggest that the regulation of CK2 activity by polyamines occur through other mechanisms, as previously described.…”