Sargiacomo M, Scherer P E, Tang Z L, Casanova J E, Lisanti M P
Whitehead Institute for Biomedical Research, Cambridge, MA 02142-1479.
Oncogene. 1994 Sep;9(9):2589-95.
Caveolae are flask-shaped micro-invaginations associated with the plasma membrane of a wide variety of cell types. Caveolin, an integral membrane component of caveolae, was first identified as the major phosphoprotein whose phosphorylation was elevated in v-Src transformed cells. As both v-Src transformation and elevated caveolin phosphorylation were dependent on membrane attachment of v-Src, it has been suggested that caveolin is a critical target in v-Src transformation. Although an increase in tyrosine phosphorylation of caveolin was evident, the increase in caveolin phosphorylation was predominantly on serine residues. In accordance with these in vivo observations, isolated caveolin-rich membrane domains undergo phosphorylation in vitro predominantly on serine and contain an unidentified serine kinase activity. Here, we have identified this serine kinase activity as a casein kinase II-like enzyme, since the phosphorylation of caveolin-rich membrane domains is stimulated and inhibited by known effectors of casein kinase II (poly-L-lysine, endogenous polyamines, and a casein kinase II inhibitor peptide), but is unaffected by modulators of other known kinases. In support of these observations, caveolin contains a consensus sequence for casein kinase II phosphorylation in its cytoplasmic N-terminal domain (Ser-88). A peptide containing this sequence inhibits the in vitro phosphorylation of caveolin-rich membrane domains, while many other peptides derived from the N-terminal domain of caveolin do not affect phosphorylation. Caveolin-rich membrane domains were also a substrate for exogenously added purified casein kinase II, but not casein kinase I. Finally, immunoblotting of these domains with an antibody directed against the alpha and alpha' subunits of casein kinase II reveals two bands with apparent molecular weights consistent with the known molecular weights of the alpha and alpha' subunits of casein kinase II. As casein kinase II appears to play a role in mitogenic signalling events and casein kinase II activators (endogenous polyamines) are required for v-Src transformation, our results may have implications for understanding the mechanism of v-Src oncogenesis.
小窝是与多种细胞类型的质膜相关的烧瓶状微内陷结构。小窝蛋白是小窝的一种整合膜成分，最初被鉴定为在v-Src转化细胞中磷酸化水平升高的主要磷蛋白。由于v-Src转化和小窝蛋白磷酸化水平升高均依赖于v-Src与膜的附着，因此有人提出小窝蛋白是v-Src转化中的关键靶点。虽然小窝蛋白酪氨酸磷酸化的增加很明显，但小窝蛋白磷酸化的增加主要发生在丝氨酸残基上。与这些体内观察结果一致，分离的富含小窝蛋白的膜结构域在体外主要在丝氨酸上发生磷酸化，并含有一种未鉴定的丝氨酸激酶活性。在这里，我们已将这种丝氨酸激酶活性鉴定为一种酪蛋白激酶II样酶，因为富含小窝蛋白的膜结构域的磷酸化受到酪蛋白激酶II的已知效应物（聚-L-赖氨酸、内源性多胺和一种酪蛋白激酶II抑制肽）的刺激和抑制，但不受其他已知激酶调节剂的影响。为支持这些观察结果，小窝蛋白在其细胞质N端结构域（Ser-88）中含有酪蛋白激酶II磷酸化的共有序列。含有该序列的肽可抑制富含小窝蛋白的膜结构域的体外磷酸化，而许多其他源自小窝蛋白N端结构域的肽则不影响磷酸化。富含小窝蛋白的膜结构域也是外源添加的纯化酪蛋白激酶II的底物，但不是酪蛋白激酶I的底物。最后，用针对酪蛋白激酶II的α和α'亚基的抗体对这些结构域进行免疫印迹，显示出两条带，其表观分子量与酪蛋白激酶II的α和α'亚基的已知分子量一致。由于酪蛋白激酶II似乎在有丝分裂信号事件中起作用，并且v-Src转化需要酪蛋白激酶II激活剂（内源性多胺），我们的结果可能对理解v-Src肿瘤发生机制具有重要意义。
