Regulation of type VI adenylyl cyclase by Snapin, a SNAP25-binding protein.
In the present study, we used the N terminus (amino acids 1 approximately 160) of type VI adenylyl cyclase (ACVI) as bait to screen a mouse brain cDNA library and identified Snapin as a novel ACVI-interacting molecule. Snapin is a binding protein of SNAP25, a component of the SNARE complex. Co-immunoprecipitation analyses confirmed the interaction between Snapin and full-length ACVI. Mutational analysis revealed that the interaction domains of ACVI and Snapin were located within amino acids 1 approximately 86 of ACVI and 33-51 of Snapin, respectively. Co-localization of ACVI and Snapin was observed in primary hippocampal neurons. Moreover, expression of Snapin specifically eliminated protein kinase C (PKC)-mediated suppression of ACVI, but not that of cAMP-dependent protein kinase (PKA) or calcium. Mutation of the potential PKC and PKA phosphorylation sites of Snapin did not affect the ability of Snapin to reverse the PKC inhibitory effect on ACVI. Phosphorylation of Snapin by PKC or PKA therefore might not be crucial for Snapin action on ACVI. In contrast, Snapin(Delta33-51), which harbors an internal deletion of amino acids 33-51 did not affect PKC-mediated inhibition of ACVI, supporting that amino acids 33-51 of Snapin comprises the ACVI-interacting region. Consistently, Snapin exerted no effect on PKC-mediated inhibition of an ACVI mutant (ACVI-DeltaA87), which lacked the Snapin-interacting region (amino acids 1-86). Snapin thus reverses its action via direct interaction with the N terminus of ACVI. Collectively, we demonstrate herein that in addition to its association with the SNARE complex, Snapin also functions as a regulator of an important cAMP synthesis enzyme in the brain.
Chou
Jui-Ling
JL
Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan.
Huang
Chuen-Lin
CL
Lai
Hsing-Lin
HL
Hung
Amos C
AC
Chien
Chen-Li
CL
Kao
Yu-Ya
YY
Chern
Yijuang
Y
D000262:Adenylyl Cyclases / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000502:physiology; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000096:biosynthesis; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D011493:Protein Kinase C / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D033921:Vesicular Transport Proteins / Q000502:physiology*
D007527:Isoenzymes; C118519:SNAPIN protein, human; C498451:Snapin protein, mouse; C498452:Snapin protein, rat; D033921:Vesicular Transport Proteins; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D000262:Adenylyl Cyclases; D002118:Calcium