Phosphorylation of dynamin II at serine-764 is associated with cytokinesis

Abstract

Calcineurin is a phosphatase that is activated at the last known stage of mitosis, abscission. Among its many substrates, it dephosphorylates dynamin II during cytokinesis at the midbody of dividing cells. However, dynamin II has several cellular roles including clathrin-mediated endocytosis, centrosome cohesion and cytokinesis. It is not known whether dynamin II phosphorylation plays a role in any of these functions nor have the phosphosites involved in cytokinesis been directly identified. We now report that dynamin II from rat lung is phosphorylated to a low stoichiometry on a single major site, Ser-764, in the proline-rich domain. Phosphorylation on Ser-764 also occurred in asynchronously growing HeLa cells and was greatly increased upon mitotic entry. Tryptic phospho-peptides isolated by TiO(2) chromatography revealed only a single phosphosite in mitotic cells. Mitotic phosphorylation was abolished by roscovitine, suggesting the mitotic kinase is cyclin-dependent kinase 1. Cyclin-dependent kinase 1 phosphorylated full length dynamin II and Glutathione-S-Transferase-tagged-dynamin II-proline-rich domain in vitro, and mutation of Ser-764 to alanine reduced proline-rich domain phosphorylation by 80%, supporting that there is only a single major phosphosite. Ser-764 phosphorylation did not affect clathrin-mediated endocytosis or bulk endocytosis using penetratin-based phospho-deficient or phospho-mimetic peptides or following siRNA depletion/rescue experiments. Phospho-dynamin II was enriched at the mitotic centrosome, but this targeting was unaffected by the phospho-deficient or phospho-mimetic peptides. In contrast, the phospho-mimetic peptide displaced endogenous dynamin II, but not calcineurin, from the midbody and induced cytokinesis failure. Therefore, phosphorylation of dynamin II primarily occurs on a single site that regulates cytokinesis downstream of calcineurin, rather than regulating endocytosis or centrosome function.

SNX9, SNX18 and SNX33 are required for progression through and completion of mitosis

“…We show that these proteins localise to the FMRs and MR within the ICB. We have recently shown that dynII locates to the FMRs (Chircop et al, 2010) and functions here in an endocytic-independent manner for abscission (Chircop et al, 2011b). Numerous proteins that locate to the MR are involved in protein and vesicular recruitment.…”

IQGAP1 is associated with nuclear envelope reformation and completion of abscission

“…We next assessed whether IQGAP1 is involved in regulating recruitment and localization of key ICB components: g-tubulin (a MR component), 53 aurora B (presence and dephosphorylation at the ICB promotes abscission), 16 calmodulin (CaM -protects aurora B from being ubiquitinated), 54 calcineurin (CaNdephosphorylates proteins such as dynamin II prior to abscission), 55,56 and PRC1 (required for microtubule polarization as well as protein recruitment to the ICB). 57 Of these, CaM directly binds IQGAP1.…”

“…56,90 IQGAP1 gel bands, each containing immunopurified protein, were excised from colloidal Coommassie Blue-stained SDS gels. The bands were destained in 3 washes of 50 mM ammonium bicarbonate in a 50% (v/v) acetonitrile solution for 15 min at 37 C. Destained bands were then dehydrated with 100% acetonitrile solution for 15 min.…”

Protein Phosphatases Drive Mitotic Exit

“…Thus, an understanding of the molecular pathways that drive mitotic exit may highlight molecular targets for the development of new anti-cancer chemotherapeutic agents. In line with this idea, a recent publication has identified the CaN substrate, dynII, as a molecular target for the treatment of cancer (Chircop et al, 2010a, Chircop et al, 2010b. Inhibitors of dynII possess anti-cancer properties due to their ability to cause cytokinesis failure and subsequent cell growth arrest or apoptotic cell death (Joshi et al, 2010).…”

“…During cytokinesis CaN locates to two 1.1 m diameter flanking midbody rings (FMRs) that reside on either side of the 1.6 m diametertubulin midbody ring (MR) within the centre of the intracellular bridge. The endocytic protein, dynamin II (dynII), is mitotically phosphorylated by Cdk1/cyclin B1 upon mitotic entry and co-localises with CaN at the FMRs during cytokinesis (Chircop et al, 2010a, Chircop et al, 2010b. CaN inhibition by CsA, dynII depletion, phospho-mimetic dynII phosphopeptides and small molecule dynamin inhibitors lead to aborted cytokinesis and multinucleation (Joshi et al, 2010, Chircop et al, 2010a, Chircop et al, 2010b, Chircop et al, 2011.…”