Deery W J, Heath J P
Department of Medicine, Baylor College of Medicine, Houston, Texas 77030.
J Cell Biol. 1993 Jul;122(1):21-37. doi: 10.1083/jcb.122.1.21.
The actin/myosin II cytoskeleton and its role in phagocytosis were examined in primary cultures of dog thyroid cells. Two (19 and 21 kD) phosphorylated light chains of myosin (P-MLC) were identified by two-dimensional gel electrophoresis of antimyosin immunoprecipitates, and were associated with the Triton X-100 insoluble, F-actin cytoskeletal fraction. Analyses of Triton-insoluble and soluble 32PO4-prelabeled protein fractions indicated that TSH (via cAMP) or TPA treatment of intact cells decreases the MLC phosphorylation state. Phosphoamino acid and tryptic peptide analyses of 32P-MLCs from basal cells showed phosphorylation primarily at threonine and serine residues; most of the [32P] appeared associated with a peptide containing sites typically phosphorylated by MLC kinase. Even in the presence of the agents which induced dephosphorylation, the phosphatase inhibitor, calyculin A, caused a severalfold increase in MLC phosphorylation at several distinct serine and threonine sites which was also associated with actomyosin and cell contraction. Phosphorylation of cell homogenate proteins or the cytoskeletal fraction with [gamma-32P]ATP indicated that Ca2+, EGTA, or trifluoperazine (TFP) has little effect on the phosphorylation of MLC. Both fluorescent phalloidin and antimyosin staining of cells showed distinct dorsal and ventral stress fiber complexes which were disrupted within 30 min by TSH and cAMP; TPA appeared to cause disruption of dorsal, and rearrangement of ventral complexes. Concomitant with MLC dephosphorylation and stress fiber disruption, TSH/cAMP, but not TPA, induced dorsal phagocytosis of latex beads. While stimulation of either A or C-kinase disrupts dorsal stress fibers and rearranges actomyosin, another event(s) mediated by A-kinase appears necessary for phagocytic activity.
在犬甲状腺细胞原代培养物中研究了肌动蛋白/肌球蛋白II细胞骨架及其在吞噬作用中的作用。通过抗肌球蛋白免疫沉淀物的二维凝胶电泳鉴定出肌球蛋白的两条（19和21kD）磷酸化轻链（P-MLC），它们与Triton X-100不溶性F-肌动蛋白细胞骨架部分相关。对Triton不溶性和可溶性32PO4预标记蛋白部分的分析表明，TSH（通过cAMP）或TPA处理完整细胞会降低MLC的磷酸化状态。对来自基础细胞的32P-MLC进行磷酸氨基酸和胰蛋白酶肽分析表明，磷酸化主要发生在苏氨酸和丝氨酸残基上；大部分[32P]似乎与一个含有通常被MLC激酶磷酸化位点的肽相关。即使在存在诱导去磷酸化的试剂的情况下，磷酸酶抑制剂花萼海绵诱癌素A也会导致在几个不同的丝氨酸和苏氨酸位点的MLC磷酸化增加几倍，这也与肌动球蛋白和细胞收缩相关。用[γ-32P]ATP对细胞匀浆蛋白或细胞骨架部分进行磷酸化表明，Ca2+、EGTA或三氟拉嗪（TFP）对MLC的磷酸化影响很小。细胞的荧光鬼笔环肽和抗肌球蛋白染色均显示出明显的背侧和腹侧应力纤维复合体，它们在30分钟内被TSH和cAMP破坏；TPA似乎导致背侧复合体的破坏和腹侧复合体的重排。与MLC去磷酸化和应力纤维破坏同时发生的是，TSH/cAMP而非TPA诱导了乳胶珠的背侧吞噬作用。虽然对A激酶或C激酶的刺激都会破坏背侧应力纤维并重新排列肌动球蛋白，但A激酶介导的另一事件似乎是吞噬活性所必需的。
