Tanaka Kenji, Okubo Yoshiko, Abe Hiroshi
Department of Biology, Chiba University, Japan.
Zoolog Sci. 2005 Sep;22(9):971-84. doi: 10.2108/zsj.22.971.
ADF/cofilin is a key regulator for actin dynamics during cytokinesis. Its activity is suppressed by phosphorylation and reactivated by dephosphorylation. Little is known, however, about regulatory mechanisms of ADF/cofilin function during formation of contractile ring actin filaments. Using Xenopus cycling extracts, we found that ADF/cofilin was dephosphorylated at prophase and telophase. In addition, constitutively active Rho GTPase induced dephosphorylation of ADF/cofilin in the egg extracts. This dephosphorylation was inhibited by Na(3)VO (4) but not by other conventional phosphatase-inhibitors. We cloned a Xenopus homologue of Slingshot phosphatase (XSSH), originally identified in Drosophila and human as an ADF/cofilin phosphatase, and raised antibody specific for the catalytic domain of XSSH. This inhibitory antibody significantly suppressed the Rho-induced dephosphorylation of ADF/cofilin in extracts, suggesting that the dephosphorylation at telophase is dependent on XSSH. XSSH bound to actin filaments with a dissociation constant of 0.4 microM, and the ADF/cofilin phosphatase activity was increased in the presence of F-actin. When latrunculin A, a G-actin-sequestering drug, was added to extracts, both Rho-induced actin polymerization and dephosphorylation of ADF/cofilin were markedly inhibited. Jasplakinolide, an actin-stabilizing drug, alone induced actin polymerization in the extracts and lead to dephosphorylation of ADF/cofilin. These results suggest that Rho-induced dephosphorylation of ADF/cofilin is dependent on the XSSH activation that is caused by increase in the amount of F-actin induced by Rho signaling. XSSH colocalized with both actin filaments and ADF/cofilin in the actin patches formed on the surface of the early cleavage furrow. Injection of inhibitory antibody blocked cleavage of blastomeres. Thus, XSSH may reorganize actin filaments through dephosphorylation and reactivation of ADF/cofilin at early stage of contractile ring formation.
ADF/丝切蛋白是胞质分裂过程中肌动蛋白动力学的关键调节因子。其活性通过磷酸化被抑制，通过去磷酸化被重新激活。然而，关于收缩环肌动蛋白丝形成过程中ADF/丝切蛋白功能的调节机制知之甚少。利用非洲爪蟾周期提取物，我们发现ADF/丝切蛋白在前期和末期发生去磷酸化。此外，组成型活性Rho GTP酶在卵提取物中诱导ADF/丝切蛋白去磷酸化。这种去磷酸化被钒酸钠抑制，但不被其他传统磷酸酶抑制剂抑制。我们克隆了弹弓磷酸酶（Slingshot phosphatase）的非洲爪蟾同源物（XSSH），最初在果蝇和人类中被鉴定为一种ADF/丝切蛋白磷酸酶，并制备了针对XSSH催化结构域的特异性抗体。这种抑制性抗体显著抑制了提取物中Rho诱导的ADF/丝切蛋白去磷酸化，表明末期的去磷酸化依赖于XSSH。XSSH以0.4微摩尔的解离常数与肌动蛋白丝结合，并且在F-肌动蛋白存在的情况下ADF/丝切蛋白磷酸酶活性增加。当向提取物中加入一种G-肌动蛋白螯合剂药物latrunculin A时，Rho诱导的肌动蛋白聚合和ADF/丝切蛋白去磷酸化均被显著抑制。一种肌动蛋白稳定药物jasplakinolide单独在提取物中诱导肌动蛋白聚合并导致ADF/丝切蛋白去磷酸化。这些结果表明，Rho诱导的ADF/丝切蛋白去磷酸化依赖于由Rho信号诱导的F-肌动蛋白量增加所导致的XSSH激活。XSSH与早期分裂沟表面形成的肌动蛋白斑中的肌动蛋白丝和ADF/丝切蛋白共定位。注射抑制性抗体阻断了卵裂球的分裂。因此，XSSH可能在收缩环形成的早期阶段通过对ADF/丝切蛋白的去磷酸化和重新激活来重组肌动蛋白丝。
