Calcium-dependent association of annexin VI, protein kinase C alpha, and neurocalcin alpha on the raft fraction derived from the synaptic plasma membrane of rat brain

A membrane microdomain enriched in cholesterol and sphingolipids or so call ed "raft" region was found to contain many signal transducing proteins such as GPI-anchored proteins, trimeric G proteins and protein tyrosine kinases , Because brain-derived raft contains two calmodulin-binding proteins, GAP- 43 and NAP-22 as the major protein components, the raft domain is assumed t o be important in the Ca2+-signaling. In this study, we analyzed protein co mponents showing Ca2+-dependent binding to the raft of synaptic plasma memb rane from rat brain. SDS-PAGE analysis of the protein components in the EGT A eluate from the raft prepared in the presence of Ca2+-ions showed the elu tion of 80 kDa, 68 kDa, 22 kDa, and 21 kDa proteins. These proteins were id entified as protein kinase Col (80 kDa) and annexin VI (68 kDa) from the pa rtial amino-acid sequencing, and neurocalcin alpha (22 kDa) and calmodulin (21 kDa) with western blotting and electrophoretic mobilities in the presen ce or absence of Ca2+ ions. Further immunoblotting experiments showed the C a2+-dependent association of conventional, but not non-conventional, subtyp es of PKC to the raft. (C) 2001 Wiley-Liss, Inc.