Rapley Joseph, Nicolàs Marta, Groen Aaron, Regué Laura, Bertran M Teresa, Caelles Carme, Avruch Joseph, Roig Joan
Department of Molecular Biology and Medical Services, Massachusetts General Hospital and Department of Medicine, Harvard Medical School, Boston, MA 02114, USA.
J Cell Sci. 2008 Dec 1;121(Pt 23):3912-21. doi: 10.1242/jcs.035360. Epub 2008 Nov 11.
Nek6 and Nercc1 (also known as Nek9) belong to the NIMA family of protein kinases. Nercc1 is activated in mitosis, whereupon it binds, phosphorylates and activates Nek6. Interference with Nek6 or Nercc1 in mammalian cells causes prometaphase-metaphase arrest, and depletion of Nercc1 from Xenopus egg extracts prevents normal spindle assembly. Herein we show that Nek6 is constitutively associated with Eg5 (also known as Kinesin-5 and Kif11), a kinesin that is necessary for spindle bipolarity. Nek6 phosphorylated Eg5 at several sites in vitro and one of these sites, Ser1033, is phosphorylated in vivo during mitosis. Whereas CDK1 phosphorylates nearly all Eg5 at Thr926 during mitosis, Nek6 phosphorylates approximately 3% of Eg5, primarily at the spindle poles. Eg5 depletion caused mitotic arrest, resulting in cells with a monopolar spindle. This arrest could be rescued by wild-type Eg5 but not by Eg5[Thr926Ala]. Despite substantial overexpression, Eg5[Ser1033Ala] rescued 50% of cells compared with wild-type Eg5, whereas an Eg5[Ser1033Asp] mutant was nearly as effective as wild type. Thus, during mitosis Nek6 phosphorylates a subset of Eg5 polypeptides at a conserved site, the phosphorylation of which is crucial for the mitotic function of Eg5.
Nek6和Nercc1（也称为Nek9）属于NIMA蛋白激酶家族。Nercc1在有丝分裂中被激活，随后它结合、磷酸化并激活Nek6。在哺乳动物细胞中干扰Nek6或Nercc1会导致前中期到中期的停滞，而从非洲爪蟾卵提取物中去除Nercc1会阻止正常纺锤体组装。在此我们表明，Nek6与Eg5（也称为驱动蛋白-5和Kif11）组成性结合，Eg5是纺锤体双极性所必需的一种驱动蛋白。Nek6在体外的几个位点磷酸化Eg5，其中一个位点Ser1033在有丝分裂期间在体内被磷酸化。虽然CDK1在有丝分裂期间在Thr926位点磷酸化几乎所有的Eg5，但Nek6主要在纺锤体极磷酸化约3%的Eg5。Eg5缺失导致有丝分裂停滞，产生具有单极纺锤体的细胞。这种停滞可以通过野生型Eg5挽救，但不能通过Eg5[Thr926Ala]挽救。尽管大量过表达，与野生型Eg5相比，Eg5[Ser1033Ala]挽救了50%的细胞，而Eg5[Ser1033Asp]突变体几乎与野生型一样有效。因此，在有丝分裂期间，Nek6在一个保守位点磷酸化一部分Eg5多肽，该位点的磷酸化对Eg5的有丝分裂功能至关重要。
