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  • Cells expressing the CDK S D mutant exhibit impaired


    Cells expressing the CDK5 S47D mutant exhibit impaired migration and enhanced proliferation: Finally, we wished to determine the functional significance of this phosphoevent. Multiple studies have implicated active CDK5 in promotion of cell migration [9,16,[21], [22], [23], [24]] and in one study the CDK5-p35 complex has been shown to be required to suppress cell proliferation [25]. Since the S47D mutation impedes the formation of a functional CDK5-p35 complex thus inhibiting its catalytic activity, we reasoned that the Quinacrine hydrochloride hydrate expressing this mutant should display retarded migration and higher proliferation. We found that such was indeed the case where in a scratch wound assay (Fig. 3), the WT and S47A CDK5 expressing cells were able to migrate and close the wound effectively. On the other hand, the cells expressing S47D CDK5 were significantly impaired in their ability to migrate and close the wound (Fig. 3). Conversely, S47D harboring cells showed a significantly higher mitotic index compared to the WT or S47A expressing cells as determined by the phospho-Histone 3 immunostaining (Fig. 4). Together, these data indicate that the inhibition of CDK5 activity by S47 phosphorylation negatively influences cell migration and promotes cell division.
    Discussion Identification of a novel phosphoregulatory mechanism to inactivate CDK5: The results presented in this study reveal a novel site-specific, phosphorylation based regulation of CDK5 activity. There are two other residues – Y15 and S159 - that have been previously implicated in phosphoregulation of CDK5 [[26], [27], [28], [29], [30], [31]]. Our study adds to the phosphoregulatory mechanisms to control CDK5 activity. The data presented here elucidate the mechanism whereby a phosphomimetic change at S47, a residue in the cyclin binding element, disrupts the binding of CDK5 to its activator resulting in failure to adopt an active conformation (Fig. 2). This potential phosphorylation based regulatory mechanism results in a loss of CDK5 activity comparable to that of the catalytically inactive D144N mutant. In fact, since the S47D mutant simulates regulation imparted by phosphorylation, it provides a more physiologically relevant mutation to control CDK5 activity. In addition, it can serve as an experimental tool to use with the D144N mutant in studies aiming to dissect the kinase activity independent roles of the CDK5-p35 complex. Notably, there is another conserved serine at position 46 (Fig. 2B) that also interfaces with the p25 binding surface (Fig. S2A). Although we did not identify S46 as a putative phosphosite in our screen, it has been previously reported in a high throughput proteomics study [32]. The phosphomimetic mutation at S46 showed a similar effect on Quinacrine hydrochloride hydrate binding to p35 (Fig. S2B) as we observed in case of S47 (Fig. 2D) suggesting that phosphorylation of either residue is sufficient in abolishing binding to p35. An alignment of the cyclin binding element from all human CDKs revealed a phosphorylatable residue (Serine in 6 members and Threonine in 14 members) at the position equivalent of S47 in 20 out of the 21 members of the family whereas position S46 is only conserved in 4 members (Fig. S3). As it is commonly observed in kinase families to adopt shared regulatory mechanisms and considering that the cyclin binding element plays a crucial role in binding of CDKs to their respective activators, it is possible that phosphorylation of S47 may represent a broad regulation strategy shared among the CDK family members. Is S47 an autophosphorylation site? Our mobility shift based western blotting assay from cellular lysates showed that the slower migrating phosphorylated bands of CDK5 appear only if the kinase is catalytically active (Fig. 1). Thus, it can be inferred that CDK5 may undergo autophosphorylation. However, an alternative explanation could be that CDK5 is phosphorylated by another kinase(s) that gets activated downstream of CDK5. The recombinant CDK5-p35 complex purified from eukaryotic insect cells (purity 84.4% as per the datasheet provided by the vendor) allowed us to capture all the phosphosites including any that might be a result of self-phosphorylation and those phosphorylated by other kinases co-purified with CDK5-p35 complex. That the latter is at least partly the case is supported by the fact that we identified Y15 in our screen. Since CDK5 is a serine/threonine kinase, the most likely explanation is that Y15 was phosphorylated by a co-purified tyrosine kinase. It also seems unlikely that S47 or the other sites we identified are autophosphorylation sites because they do not satisfy the minimum consensus site requirement for CDK5 i.e. a serine or threonine residue followed by a proline (S/T-P motif) [33]. Nevertheless, we cannot completely rule out the possibility of autophosphorylation as kinases can phosphorylate non-consensus sites.