Ben-Ya'acov Ami, Ravid Shoshana
Department of Biochemistry, Institute of Medical Sciences, Hadassah Medical School, Hebrew University, Jerusalem 91120, Israel.
J Biol Chem. 2003 Oct 10;278(41):40032-40. doi: 10.1074/jbc.M306948200. Epub 2003 Jul 21.
Epidermal growth factor (EGF) stimulation of prostate metastatic tumor cells results in transient phosphorylation and cellular localization of non-muscle myosin heavy chain II-B (NMHC II-B) with kinetics similar to those seen in chemotaxis. We demonstrate that expression of 18- and 72-kDa fragments derived from the NMHC II-B C terminus that contain EGF-dependent NMHC II-B phosphorylation sites serve as dominant-negative mutations for EGF-dependent NMHC II-B phosphorylation and localization. Both fragments inhibited the EGF-dependent phosphorylation by competing with NMHC II-B on the myosin heavy chain kinase. However, only expression of the 72-kDa fragment resulted in cells with abnormalities in cell shape, focal adhesions, and chemotaxis. We found that the 72-kDa (but not 18-kDa) fragment is capable of self-assembly. To our knowledge, these results provide the first strong evidence that EGF-dependent NMHC II-B phosphorylation is required for the cellular localization of NMHC II-B and that NMHC II-B is required for normal cell attachment and for chemotactic response.
表皮生长因子（EGF）对前列腺转移肿瘤细胞的刺激导致非肌肉肌球蛋白重链II - B（NMHC II - B）的瞬时磷酸化和细胞定位，其动力学与趋化作用中观察到的相似。我们证明，源自NMHC II - B C末端的18 kDa和72 kDa片段的表达，这些片段包含依赖于EGF的NMHC II - B磷酸化位点，可作为依赖于EGF的NMHC II - B磷酸化和定位的显性负性突变。两个片段都通过在肌球蛋白重链激酶上与NMHC II - B竞争来抑制依赖于EGF的磷酸化。然而，只有72 kDa片段的表达导致细胞在细胞形状、粘着斑和趋化性方面出现异常。我们发现72 kDa（而非18 kDa）片段能够自我组装。据我们所知，这些结果首次有力证明，依赖于EGF的NMHC II - B磷酸化是NMHC II - B细胞定位所必需的，并且NMHC II - B是正常细胞附着和趋化反应所必需的。
