Yi Kexi, Buttery Shawnna M, Stewart Murray, Roberts Thomas M
Department of Biological Science, Florida State University, Tallahassee, FL 32306, USA.
Mol Biol Cell. 2007 May;18(5):1816-25. doi: 10.1091/mbc.e06-08-0741. Epub 2007 Mar 7.
Leading edge protrusion in the amoeboid sperm of Ascaris suum is driven by the localized assembly of the major sperm protein (MSP) cytoskeleton in the same way that actin assembly powers protrusion in other types of crawling cell. Reconstitution of this process in vitro led to the identification of two accessory proteins required for MSP polymerization: an integral membrane phosphoprotein, MSP polymerization-organizing protein (MPOP), and a cytosolic component, MSP fiber protein 2 (MFP2). Here, we identify and characterize a 34-kDa cytosolic protein, MSP polymerization-activating kinase (MPAK) that links the activities of MPOP and MFP2. Depletion/add-back assays of sperm extracts showed that MPAK, which is a member of the casein kinase 1 family of Ser/Thr protein kinases, is required for motility. MPOP and MPAK comigrated by native gel electrophoresis, coimmunoprecipitated, and colocalized by immunofluorescence, indicating that MPOP binds to and recruits MPAK to the membrane surface. MPAK, in turn, phosphorylated MFP2 on threonine residues, resulting in incorporation of MFP2 into the cytoskeleton. Beads coated with MPAK assembled a surrounding cloud of MSP filaments when incubated in MPAK-depleted sperm extract, but only when supplemented with detergent-solubilized MPOP. Our results suggest that interactions involving MPOP, MPAK, and MFP2 focus MSP polymerization to the plasma membrane at the leading edge of the cell thereby generating protrusion and minimizing nonproductive filament formation elsewhere.
猪蛔虫变形虫样精子的前缘突出是由主要精子蛋白（MSP）细胞骨架的局部组装驱动的，其方式与肌动蛋白组装驱动其他类型爬行细胞的突出相同。在体外对这一过程进行重构，从而鉴定出MSP聚合所需的两种辅助蛋白：一种整合膜磷蛋白，即MSP聚合组织蛋白（MPOP），以及一种胞质成分，即MSP纤维蛋白2（MFP2）。在这里，我们鉴定并表征了一种34 kDa的胞质蛋白，即MSP聚合激活激酶（MPAK），它连接了MPOP和MFP2的活性。对精子提取物进行的缺失/回补试验表明，作为丝氨酸/苏氨酸蛋白激酶酪蛋白激酶1家族成员的MPAK是运动所必需的。MPOP和MPAK通过天然凝胶电泳共迁移、共免疫沉淀，并通过免疫荧光共定位，表明MPOP与MPAK结合并将其招募到膜表面。反过来，MPAK使MFP2的苏氨酸残基磷酸化，导致MFP2整合到细胞骨架中。当在缺乏MPAK的精子提取物中孵育时，包被有MPAK的珠子组装了一团围绕的MSP细丝云，但只有在补充了去污剂溶解的MPOP时才会如此。我们的结果表明，涉及MPOP、MPAK和MFP2的相互作用将MSP聚合聚焦到细胞前缘的质膜上，从而产生突出并将其他地方非生产性细丝形成最小化。
