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 called "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 to be important in the Ca(2+)-signaling. In this study, we analyzed protein components showing Ca(2+)-dependent binding to the raft of synaptic plasma membrane from rat brain. SDS-PAGE analysis of the protein components in the EGTA eluate from the raft prepared in the presence of Ca(2+)-ions showed the elution of 80 kDa, 68 kDa, 22 kDa, and 21 kDa proteins. These proteins were identified as protein kinase C alpha (80 kDa) and annexin VI (68 kDa) from the partial amino-acid sequencing, and neurocalcin alpha (22 kDa) and calmodulin (21 kDa) with western blotting and electrophoretic mobilities in the presence or absence of Ca(2+) ions. Further immunoblotting experiments showed the Ca(2+)-dependent association of conventional, but not non-conventional, subtypes of PKC to the raft.
Orito
A
A
Department of Biotechnology, Faculty of Textile Sciences, Kyoto Institute of Technology, Kyoto, Japan.
Kumanogoh
H
H
Yasaka
K
K
Sokawa
J
J
Hidaka
H
H
Sokawa
Y
Y
Maekawa
S
S
D000818:Animals; D017317:Annexin A6 / Q000378:metabolism*; D001921:Brain / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism*; D021962:Membrane Microdomains / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D051596:Neurocalcin; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D051381:Rats; D044169:Receptors, Calcium-Sensing; D013570:Synaptic Membranes / Q000378:metabolism*
D017317:Annexin A6; D002135:Calcium-Binding Proteins; D007527:Isoenzymes; D009419:Nerve Tissue Proteins; D051596:Neurocalcin; D044169:Receptors, Calcium-Sensing; C496690:Vsnl1 protein, rat; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D002118:Calcium