Department of Chemistry and Biochemistry, Queens College, The City University of New York, NY 11367, USA.
Cell Signal. 2010 Jul;22(7):1097-103. doi: 10.1016/j.cellsig.2010.03.003. Epub 2010 Mar 6.
Phosphorylation of myristoylated alanine-rich C-kinase substrate (MARCKS) by protein kinase C alpha (PKC alpha) is known to trigger its release from the plasma membrane/cytoskeleton into the cytoplasm, thereby promoting actin reorganization during migration. This study shows that once released into the cytoplasm, phosphoMARCKS directly promotes motility of melanoma cells. Aggressively motile B16 F10 mouse melanoma cells express high levels of phosphoMARCKS, whereas in weakly motile B16 F1 cells it is undetectable. Following treatment with okadaic acid (OA) (a protein phosphatase inhibitor), F1 cells exhibited a dramatic increase in phosphoMARCKS that was co-incident with a 5-fold increase in motility. Both MARCKS phosphorylation and motility were substantially decreased when prior to OA addition, MARCKS expression was knocked out by a MARCKS-specific shRNA, thereby implicating MARCKS as a major component of the motility pathway. Decreased motility and phosphoMARCKS levels in OA-treated cells were observed with a PKC inhibitor (calphostin C), thus indicating that PKC actively phosphorylates MARCKS in F1 cells but that this reaction is efficiently reversed by protein phosphatases. The mechanistic significance of phosphoMARCKS to motility was further established with a pseudo-phosphorylated mutant of MARCKS-GFP in which Asp residues replaced Ser residues known to be phosphorylated by PKC alpha. This mutant localized to the cytoplasm and engendered three-fold higher motility in F1 cells. Expression of an unmyristoylated, phosphorylation-resistant MARCKS mutant that localized to the cytoplasm, blocked motility by 40-50% of both OA-stimulated F1 cells and intrinsically motile F10 cells. These results demonstrate that phosphoMARCKS contributes to the metastatic potential of melanoma cells, and reveal a previously undocumented signaling role for this cytoplasmic phospho-protein.
蛋白激酶 Cα(PKCα)对豆蔻酰化丙氨酸丰富的蛋白激酶 C 底物(MARCKS)的磷酸化作用,已知会触发其从质膜/细胞骨架释放到细胞质中,从而促进迁移过程中的肌动蛋白重组。本研究表明,一旦释放到细胞质中,磷酸化 MARCKS 可直接促进黑色素瘤细胞的迁移。侵袭性强的 B16 F10 小鼠黑色素瘤细胞表达高水平的磷酸化 MARCKS,而在迁移能力较弱的 B16 F1 细胞中则无法检测到。用 okadaic acid(OA)(一种蛋白磷酸酶抑制剂)处理后,F1 细胞中磷酸化 MARCKS 的水平显著增加,同时迁移能力增加了 5 倍。在 OA 加入之前,通过 MARCKS 特异性 shRNA 敲除 MARCKS 表达,MARCKS 磷酸化和迁移都显著降低,这表明 MARCKS 是迁移途径的主要组成部分。在用 PKC 抑制剂(calphostin C)处理的 OA 处理的细胞中,观察到迁移能力和磷酸化 MARCKS 水平降低,这表明 PKC 在 F1 细胞中积极磷酸化 MARCKS,但该反应可被蛋白磷酸酶有效逆转。用 MARCKS-GFP 的拟磷酸化突变体进一步证实了磷酸化 MARCKS 对迁移的重要性,该突变体中的 Asp 残基取代了已知被 PKCα磷酸化的 Ser 残基。该突变体定位于细胞质中,使 F1 细胞的迁移能力增加了三倍。表达一种未豆蔻酰化、磷酸化抗性的 MARCKS 突变体,该突变体定位于细胞质中,可使 OA 刺激的 F1 细胞和固有迁移能力的 F10 细胞的迁移能力降低 40-50%。这些结果表明,磷酸化 MARCKS 有助于黑色素瘤细胞的转移潜力,并揭示了这种细胞质磷酸化蛋白以前未被记录的信号作用。