The linkage between β1 integrin and the actin cytoskeleton is differentially regulated by tyrosine and serine/threonine phosphorylation of β1 integrin in normal and cancerous human breast cells

BACKGROUND: Structural requirements for the β1 integrin functions in cell adhesion, spreading and signaling have been well documented mainly for fibroblasts. In this study, we examined the reason for the reduced surface expression of β1 integrin in human breast cancer MCF-7 cells compared to normal human breast epithelial (HBE) cells, both of which adhered to collagen type IV. RESULTS: The β1 integrin immunoprecipitates from either HBE or MCF-7 cells involved α-actinin while actin coprecipitated with β1 integrin from HBE cells but not from MCF-7 cells. Immunoblotting using the anti-phosphotyrosine (PY) antibody indicated the phosphorylation of β1 integrin at least at tyrosine in both cells. Dephosphorylation of β1 integrin from HBE cells by protein tyrosine phosphatase (PTP), but not by protein serine/threonine phosphatase (PP), caused dissociation of actin from β1 integrin, although dephosphorylation of it from MCF-7 cells by either PTP or PP caused association of the two proteins. In MCF-7 cells β1 integrin coprecipitated doublet of proteins having the Ca(2+)/calmodulin-dependent protein kinase (CaMK) II activity that was susceptible to KN-62, a specific inhibitor of CaMKII. CONCLUSION: The results suggest that β1 integrin is tyrosine phosphorylated and links with actin via α-actinin in HBE cells but prevented from linking with actin in MCF-7 cells by phosphorylation at both tyrosine and serine/threonine of β1 integrin which forms a complex with α-actinin and CaMKII. Thus the linkage formation of β1 integrin with actin may be differentially regulated by its tyrosine and serine/threonine phosphorylation in normal HBE cells and breast cancer MCF-7 cells.

背景：β1整合素（β1 integrin）在细胞黏附、铺展及信号传导中的功能所需的结构基础，主要针对成纤维细胞已有较为详尽的研究记载。本研究针对两种均能黏附于IV型胶原的细胞——正常人乳腺上皮（HBE）细胞与人乳腺癌MCF-7细胞，探究了后者β1整合素表面表达量较前者降低的原因。

结果：免疫沉淀实验结果显示，HBE与MCF-7细胞的β1整合素免疫沉淀物中均含有α-辅肌动蛋白（α-actinin）；但从HBE细胞中，肌动蛋白（actin）可与β1整合素共沉淀，而MCF-7细胞中未检测到此现象。采用抗磷酸化酪氨酸（anti-phosphotyrosine, PY）抗体进行免疫印迹实验，结果表明两种细胞中的β1整合素至少在酪氨酸位点发生了磷酸化修饰。对HBE细胞来源的β1整合素，使用蛋白酪氨酸磷酸酶（protein tyrosine phosphatase, PTP）进行去磷酸化处理，可使肌动蛋白与β1整合素解离，而蛋白丝氨酸/苏氨酸磷酸酶（protein serine/threonine phosphatase, PP）则无此效果；但针对MCF-7细胞来源的β1整合素，无论使用PTP还是PP进行去磷酸化处理，均能促进两种蛋白的结合。在MCF-7细胞中，与β1整合素共沉淀的双条带蛋白具有Ca²⁺/钙调蛋白依赖性蛋白激酶（Ca²⁺/calmodulin-dependent protein kinase, CaMK）II活性，且该活性可被CaMKII特异性抑制剂KN-62所抑制。

结论：本研究结果表明，在HBE细胞中，β1整合素发生酪氨酸磷酸化，并通过α-辅肌动蛋白与肌动蛋白结合；而在MCF-7细胞中，β1整合素的酪氨酸与丝氨酸/苏氨酸位点均发生磷酸化，且该整合素与α-辅肌动蛋白及CaMKII形成复合物，从而阻断了其与肌动蛋白的结合。由此可见，在正常人乳腺上皮细胞与乳腺癌MCF-7细胞中，β1整合素与肌动蛋白的连接形成，可通过其酪氨酸与丝氨酸/苏氨酸磷酸化状态受到差异性调控。