Mutz Diana, Weise Christoph, Mechai Nadja, Hofmann Werner, Horstkorte Rüdiger, Brüning Gerold, Danker Kerstin
Institut für Molekularbiologie und Biochemie, Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Berlin-Dahlem, Germany.
J Neurosci Res. 2006 Dec;84(8):1759-70. doi: 10.1002/jnr.21078.
Integrin alpha3beta1 is a receptor for the extracellular matrix component laminin 5. To elucidate possible signaling pathways induced by integrin alpha3beta1, we looked for proteins that interact with the cytoplasmic part of the alpha3A integrin subunit. We identified several multifunctional proteins by affinity chromatography and subsequent MALDI-TOF-MS and focused on the inhibitor 1 of serine/threonine phosphatase PP2A (I1PP2A, synonym: lanp) which also plays a role during the development of the mouse cerebellum. I1PP2A/lanp colocalizes with the alpha3A integrin subunit in differentiated PC12 cells in the cell body and in neurites as well as in Purkinje cells of mouse cerebellum. Overexpression of GFP-I1PP2A/lanp in PC12 cells leads to markedly reduced neurite length on laminin 5 after induction with nerve growth factor. By affinity chromatography the protein phosphatase PP1 can also be identified as a alpha3A/cyto-binding protein. PP1 and integrin alpha3beta1 can be pulled down by GST-I1PP2A/lanp from cell lysates of differentiated and undifferentiated PC12 cells. The phosphatase binds to the cytoplasmic membrane-proximal conserved GFFKR motif of the alpha integrin subunit, whereas I1PP2A/lanp requires a longer sequence for binding. PP1 but not PP2A is able to dephosphorylate precipitated integrin alpha3beta1 in vitro. Furthermore, PP1 releases phosphate from T1046 of phosphopeptides that mimic the phosphorylation consensus sequence in the cytoplasmic part of the alpha3A integrin subunit. These data suggest that I1PP2A/lanp forms a complex with PP1 and the alpha3A integrin subunit and might possibly regulate the phosphorylation status of integrin alpha3beta1 and/or integrin downstream targets.
整合素α3β1是细胞外基质成分层粘连蛋白5的受体。为了阐明整合素α3β1诱导的可能信号通路，我们寻找与α3A整合素亚基细胞质部分相互作用的蛋白质。我们通过亲和层析以及随后的基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）鉴定了几种多功能蛋白质，并聚焦于丝氨酸/苏氨酸磷酸酶PP2A的抑制剂1（I1PP2A，同义词：lanp），它在小鼠小脑发育过程中也发挥作用。I1PP2A/lanp与α3A整合素亚基在分化的PC12细胞的细胞体、神经突以及小鼠小脑的浦肯野细胞中共定位。在PC12细胞中过表达绿色荧光蛋白（GFP）-I1PP2A/lanp会导致在用神经生长因子诱导后，细胞在层粘连蛋白5上的神经突长度显著缩短。通过亲和层析，蛋白磷酸酶PP1也可被鉴定为α3A/细胞结合蛋白。从分化和未分化的PC12细胞裂解物中，PP1和整合素α3β1可被谷胱甘肽S-转移酶（GST）-I1PP2A/lanp拉下。该磷酸酶与α整合素亚基的细胞质膜近端保守GFFKR基序结合，而I1PP2A/lanp结合需要更长的序列。PP1而非PP2A能够在体外使沉淀的整合素α3β1去磷酸化。此外，PP1从模拟α3A整合素亚基细胞质部分磷酸化共有序列的磷酸肽的T1046位点释放磷酸盐。这些数据表明，I1PP2A/lanp与PP1和α3A整合素亚基形成复合物，可能调节整合素α3β1和/或整合素下游靶点的磷酸化状态。
