Here we have explored the mechanisms of Emi

Here, we have explored the mechanisms of Emi2 inhibition by Cdk1 (as well as Emi2 upregulation by Mos) using Xenopus egg extracts. Our results show that multiple distinct kinases, including CAY10499 synthesis B1-Cdk1 itself, bind to the N-terminal Cdk1 sites of Emi2 and phosphorylate other critical sites to destabilize and inactivate Emi2. Mos/Rsk-recruited PP2A, identified here as PP2A-B56β/ε, keeps preferentially dephosphorylating the critical inhibitory sites of Emi2. Thus, Emi2 stability and activity are dynamically regulated by Emi2-bound multiple kinases and PP2A phosphatase, providing new insight into the mechanisms of Emi2 regulation in Meta-II arrest of vertebrate eggs. In addition, our data seem to provide general implications for Cdk1 substrate phosphorylation motifs in M phase regulation.
Results
Discussion Our results clarify precise, dynamic control mechanisms of Emi2 stability and activity in Meta-II arrest of Xenopus eggs, and, together with recently published data, show how Meta-II arrest is robustly maintained (Figure 7). In addition, our data seem to suggest a general role for Cdk1 substrate phosphorylation motifs in M phase regulation.
Experimental Procedures Biochemical reagents, cDNAs, in vitro transcription/translation, antibodies, immunoblotting, in vitro kinase assays (including GST-Emi2 peptide fusion proteins), and pulldown assays are described in the Supplemental Information.
Acknowledgments
Introduction Members of the serine/threonine specific protein kinase CK1 were among the first protein kinases having been described in the literature [1], [2]. They are ubiquitously expressed, evolutionarily highly conserved and found in all eukaryotes ranging from yeast to humans [3], [4]. At present, seven mammalian CK1 isoforms have been characterised (α, β, γ1, γ2, γ3, δ, ε) [5], [6], [7], [8], [9] and more than 140 in vitro and in vivo substrates of CK1 are listed (reviewed in Ref. 4). CK1 plays important regulative roles in various cellular processes, including proliferation, apoptosis, cell differentiation, circadian rhythm, chromosome segregation and membrane trafficking [4], [10]. Deregulation of CK1 expression and activity contributes to the pathogenesis of neurodegenerative diseases and cancer. Therefore, CK1 isoforms are interesting new targets for the development of CK1 specific inhibitors and biological tools to inhibit CK1 activity which influences the regulation of microtubule dynamics and the spindle apparatus. It has been shown that CK1δ phosphorylates α-, β- and γ-tubulin in vitro and that CK1δ specifically interacts with the trans Golgi network, COPI positive vesicles, and centrosomes in interphase cells [11], [12], [13], [14]. Moreover CK1δ is also associated with granular particles that are associated with microtubules [11], [15], and recently, it was shown that siRNA induced knockdown of CK1δ inhibits microtubule nucleation at the Golgi apparatus [16]. CK1δ is recruited to the spindle apparatus during mitosis [17] and its association is significantly enhanced upon DNA-damage induced by camptothecin, etoposide or γ-irradiation [15]. CK1δ inhibition by specific small molecule inhibitors led to structural alterations of centrosomes, the formation of multipolar spindles, and inhibition of mitosis [17], [18]. Furthermore, at lower concentrations of CK1 specific inhibitors, mitotic spindle dynamics was affected leading to cell cycle arrest and, depending on the cellular background, to apoptosis in a dose-dependent manner [11], [19]. In order to maintain genomic stability under conditions of genotoxic stress, CK1δ regulates microtubule and spindle dynamics by phosphorylation of Sid4 thereby delaying cytokinesis [20] as well as by phosphorylation of MAP4, MAP1A, tau, and Stathmin [4], [10], [15], [21].