The finding that protein kinase CK2α was posttranslationally modified by O-GlcNAc on a site near a known Cdk-phosphorylation site led us to explore how these modifications may impact CK2 regulation. Expressed protein ligation is a strategy that was well suited for this task, since the modification sites are near the C-terminus of the recombinant protein. We provide evidence that Thr344 phosphorylation and Ser347 O-GlcNAcylation of CK2α transmit reciprocal information important for cell signaling networks. Thr344 phosphorylation enhances the stability of CK2α by Pin1 interaction and can also shape the pattern of protein substrates targeted by CK2. In contrast, it appears that Ser347 O-GlcNAcylation of CK2α may limit its cellular stability by altering Thr344 phosphorylation, and this sugar residue can further induce a distinct pattern of protein substrates phosphorylated by the kinase. Even though we did not see differences in stability between the unmodified and S-GlcNAc-Ser347 CK2α proteins in the microinjection experiments at the time points analyzed, we did observe a modest but reproducible reduction in endogenous CK2α levels when treating with TMG for a longer period. These results may differ since it is presumed that only a minute fraction of the unmodified microinjected protein is phosphorylated in cells during the course of the experiment.
Phosphorylation-dependent interactions with Pin1 have been shown to be stabilizing for other proteins including Nanog40
. How Pin1 binding can stabilize CK2 from proteasome-mediated destruction is not yet settled, although one possible mechanism is that the Pin1 complex prevents ubiquitination by sequestration from a requisite E3 ligase42
. A consequence of our findings is that CK2α levels can spike in cells during the period of mitosis, which might lead to a special set of phosphorylation events. In addition, the function of the different patterns of substrate phosphorylation conferred by Cdk-mediated modification of CK2α and Pin1 interaction might have important roles in mitotic regulation.
The ability of pThr344 and S-GlcNAc347 to influence changes in phosphorylation by CK2 via direct contacts with the purified protein substrates summarized in is a noteworthy finding. PTM regulation of the function of kinases is typically associated with general activation or inhibition of a kinase, or to serve as a specific recruiting site for phosphoprotein binding adaptor molecules like SH2 domain or 14-3-3 domain containing proteins. Such specific PTM binding domains are not readily apparent in the CK2 substrates in , so the mechanism for specificity will be an interesting structural biology challenge for future studies. The implications of the particular CK2 substrate interactions that we identified in and Supplementary Table 4
suggest a wide range of biologically processes being affected. For example, CK2 catalyzed phosphorylation of eEF2 kinase and WASL (N-WASP), inhibited by CK2 phosphorylation on Thr344, could influence translation43
and cytoskeletal dynamics44,45
, respectively. In contrast, pThr344-CK2 is more active as a kinase toward histone chaperone NAP1L3, which could modulate chromatin dynamics46
. The reversible kinetics of CK2 PTMs in vivo, not captured here using fixed forms of CK2s in the microarray studies, could indeed create a mosaic of complex cellular effects on the proteome.
The marrying of expressed protein ligation to generate site-specific and stoichiometrically modified forms of a protein kinase with protein microarray phosphorylation assays, performed here for the first time, can help illuminate distinct connections in signaling cascades that would be very difficult to discern using other biochemical approaches. While mass-spectrometric based phosphoproteomics47
using inhibitors or chemical complementation48
in cellular assays is powerful, it cannot readily provide insights into the substrate-altering behavior of specific PTMs on a kinase. As shown in our study, the common mutagenesis phospho mimic approach, Thr to Glu, fails to replicate the properties of pThr344 in CK2α in Pin1 interaction or in selective phosphorylation of NAP1L3. Moreover, there is no natural amino acid residue that can even be considered a plausible mimic of an O-GlcNAc moiety.
In summary, the complex regulation of CK2 by competing phosphorylation and O-GlcNAcylation provides a new framework for understanding this key signaling enzyme. This study also provides a comprehensive approach to investigating how protein PTMs can mediate multiple actions in a cellular network.