Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, New York 14263, USA.
J Biol Chem. 2010 Feb 12;285(7):4578-86. doi: 10.1074/jbc.M109.073494. Epub 2009 Dec 15.
SSeCKS/Gravin/AKAP12 ("SSeCKS") encodes a cytoskeletal protein that regulates G(1) --> S progression by scaffolding cyclins, protein kinase C (PKC) and PKA. SSeCKS is down-regulated in many tumor types including prostate, and when re-expressed in MAT-LyLu (MLL) prostate cancer cells, SSeCKS selectively inhibits metastasis by suppressing neovascularization at distal sites, correlating with its ability to down-regulate proangiogenic genes including Vegfa. However, the forced re-expression of VEGF only rescues partial lung metastasis formation. Here, we show that SSeCKS potently inhibits chemotaxis and Matrigel invasion, motility parameters contributing to metastasis formation. SSeCKS suppressed serum-induced activation of the Raf/MEK/ERK pathway, resulting in down-regulation of matrix metalloproteinase-2 expression. In contrast, SSeCKS had no effect on serum-induced phosphorylation of the Src substrate, Shc, in agreement with our previous data that SSeCKS does not inhibit Src kinase activity in cells. Invasiveness and chemotaxis could be restored by the forced expression of constitutively active MEK1, MEK2, ERK1, or PKCalpha. SSeCKS suppressed phorbol ester-induced ERK1/2 activity only if it encoded its PKC binding domain (amino acids 553-900), suggesting that SSeCKS attenuates ERK activation through a direct scaffolding of conventional and/or novel PKC isozymes. Finally, control of MLL invasiveness by SSeCKS is influenced by the actin cytoskeleton: the ability of SSeCKS to inhibit podosome formation is unaffected by cytochalasin D or jasplakinolide, whereas its ability to inhibit MEK1/2 and ERK1/2 activation is nullified by jasplakinolide. Our findings suggest that SSeCKS suppresses metastatic motility by disengaging activated Src and then inhibiting the PKC-Raf/MEK/ERK pathways controlling matrix metalloproteinase-2 expression and podosome formation.
SSeCKS/Gravin/AKAP12（“SSeCKS”）编码一种细胞骨架蛋白，通过支架细胞周期蛋白、蛋白激酶 C（PKC）和 PKA，调节 G1 到 S 期的进展。SSeCKS 在许多肿瘤类型中下调，包括前列腺癌，当在 MAT-LyLu（MLL）前列腺癌细胞中重新表达时，SSeCKS 通过抑制远端部位的新生血管生成选择性抑制转移，这与其下调包括 Vegfa 在内的促血管生成基因的能力相关。然而，VEGF 的强制重新表达仅挽救了部分肺转移的形成。在这里，我们表明 SSeCKS 强烈抑制趋化性和 Matrigel 侵袭，这是转移形成的运动参数。SSeCKS 抑制了血清诱导的 Raf/MEK/ERK 通路的激活，导致基质金属蛋白酶-2 表达的下调。相比之下，SSeCKS 对血清诱导的Src 底物 Shc 的磷酸化没有影响，这与我们之前的数据一致，即 SSeCKS 不会抑制细胞中的Src 激酶活性。通过强制表达组成型激活的 MEK1、MEK2、ERK1 或 PKCalpha，可恢复侵袭性和趋化性。只有当 SSeCKS 编码其 PKC 结合结构域（氨基酸 553-900）时，它才能抑制佛波醇酯诱导的 ERK1/2 活性，这表明 SSeCKS 通过直接支架构象常规和/或新型 PKC 同工酶来减弱 ERK 激活。最后，SSeCKS 对 MLL 侵袭性的控制受肌动蛋白细胞骨架的影响：SSeCKS 抑制破骨细胞形成的能力不受细胞松弛素 D 或 jasplakinolide 的影响，而其抑制 MEK1/2 和 ERK1/2 激活的能力则被 jasplakinolide 消除。我们的研究结果表明，SSeCKS 通过分离激活的Src 并抑制控制基质金属蛋白酶-2 表达和破骨细胞形成的 PKC-Raf/MEK/ERK 途径来抑制转移性运动。
