Direct Regulation of Microtubule Dynamics by Protein Kinase CK2

Abstract

Microtubule dynamics is essential for many vital cellular processes such as morphogenesis and motility. Protein kinase CK2 is a ubiquitous protein kinase that is involved in diverse cellular functions. CK2 holoenzyme is composed of two catalytic ␣ or ␣ subunits and two regulatory ␤ subunits. We show that the ␣ subunit of CK2 binds directly to both microtubules and tubulin heterodimers. CK2 holoenzyme but neither of its individual subunits exhibited a potent effect of inducing microtubule assembly and bundling. Moreover, the polymerized microtubules were strongly stabilized by CK2 against cold-induced depolymerization. Interestingly, the kinase activity of CK2 is not required for its microtubule-assembling and stabilizing function because a kinase-inactive mutant of CK2 displayed the same microtubule-assembling activity as the wild-type protein.Knockdown of CK2␣/␣ in cultured cells by RNA interference dramatically destabilized their microtubule networks, and the destabilized microtubules were readily destructed by colchicine at a very low concentration. Further, over-expression of chicken CK2␣ or its kinaseinactive mutant in the endogenous CK2␣/␣-depleted cells fully restored the microtubule resistance to the low dose of colchicine. Taken together, CK2 is a microtubule-associated protein that confers microtubule stability in a phosphorylation-independent manner. Protein kinase CK2 (formerly known as casein kinase 2) is ubiquitously expressed and highly conserved in eukaryotic cells (1-4). It comprises two catalytic ␣ or ␣Ј subunits and two regulatory ␤ subunits to form a heterotetrameric structure in which the two ␤ subunits dimerize to link the two ␣ or ␣Ј subunits (5). As a protein serine/threonine kinase, CK2 has a very broad phosphorylation spectrum, and over 300 protein substrates of CK2 have been identified to date (6). A number of studies have indicated that CK2 is involved in a wide variety of cellular processes including cell cycle, apoptosis, transcriptional regulation, and signal transduction (1, 3, 6). CK2 is instrumental and necessary for promoting cell survival (3, 7). Disruption of genes encoding both of the catalytic subunits of CK2 is synthetic lethal in fission yeast (8,9). Similarly, it is embryonic lethal when CK2␤ is knocked down in Caenorhabditis elegans by RNA interference or in mice by gene disruption, reminiscent of an essential role of CK2␤ during embryonic development and organogenesis (10, 11). Hence, production of both the ␣ and ␤ subunits of CK2 appears to be mandatory for cell viability.A few lines of evidence have lead to implication that CK2 might be involved in the regulation of microtubule cytoskeleton reorganization (12-14). CK2 was localized to microtubule structures such as the mitotic spindle of dividing cells and was found to associate with the cold-stable fraction of microtubules from the rat brain (14, 15). More recently, the ␣ and ␣Ј subunits were shown to bind tubulin in a far Western assay (16). Further, CK2 is able to phosphorylate a number of microtubule elements, in...

Identification of casein kinase 1, casein kinase 2, and cAMP-dependent protein kinase-like activities in Trypanosoma evansi

“…Also, coimmunoprecipitation and in vitro binding studies have evidenced that CK2 is strongly associated to tubulin in mammalian cell extracts (Faust et al 1999). More recently, chicken CK2 has been identified as a structural MAP that stabilizes microtubules and mediates microtubule dynamics (Lim et al 2004). However, the CK2 microtubule assembling and stabilizing function was independent of its kinase activity, since a kinase-inactive mutant of CK2 displayed the same microtubule assembling activity as the wild-type protein (Lim et al 2004).…”

“…More recently, chicken CK2 has been identified as a structural MAP that stabilizes microtubules and mediates microtubule dynamics (Lim et al 2004). However, the CK2 microtubule assembling and stabilizing function was independent of its kinase activity, since a kinase-inactive mutant of CK2 displayed the same microtubule assembling activity as the wild-type protein (Lim et al 2004). Interestingly, tubulin and the CK2 responsible for its phosphorylation have also been shown to be associated in T. cruzi (Casas et al Fig.…”

Protein Kinase CK2 Is an Inhibitor of the Neuronal Cdk5 Kinase

“…Recombinant proteins tagged with GST 1 or His 6 were expressed in bacteria and purified as described in earlier reports (30,31). Escherichia coli BL21(DE3) was used for protein expression from pGEX and pET32a constructs, and E. coli M15 was the expression host for the pQE30 construct.…”

“…Transient transfection was carried out using Effectene (Qiagen) for 24 h. CK2␣ and CK2␤ were cloned into the bicistronic vector pBudCE4.1 (Invitrogen) for co-expression of the two proteins. The siRNA sequence used against human CK2␣/␣Ј was described in an earlier report (30). 20 M CK2␣/␣Ј siRNA or a scrambled siRNA sequence (Dharmacon) was applied into transfections using the TransIT-TKO reagent (Mirus).…”

“…CK2␣ and its kinase-dead mutant CK2␣K68A were cloned into pET32a, and human CK2␤ was cloned into pQE30 (30).…”