A central kinase domain of type I phosphatidylinositol phosphate kinases is sufficient to prime exocytosis: isoform specificity and its underlying mechanism.
Exocytosis, a critical process for neuronal communication and hormonal regulation, involves several distinct steps including MgATP-dependent priming (which involves the synthesis of phosphatidylinositol 4,5-bisphosphate). Type I phosphatidylinositol phosphate kinases (PIPKIs) were purified biochemically as a priming factor. PIPKI consists of three domains: the N-terminal region, the central kinase domain, and the C-terminal region. Three isoforms (alpha, beta, and gamma) of PIPKI have been identified, and each is alternatively spliced at the C-terminal region. In the present study, we conducted a structure/function analysis of PIPKIs in the priming of exocytosis, and we found that recombinant PIPKIalpha and PIPKIgamma had priming activity. However, an unexpected finding of these results was that PIPKIbeta did not prime exocytosis. The N- or C-terminal region of PIPKIalpha and PIPKIgamma was not required for priming, which indicates that the central kinase domain is sufficient for this process. Alternative splicing in each isoform did not affect the isoform specificity in priming. Priming activity by isoforms is strongly correlated with their phosphatidylinositol phosphate kinase activity because PIPKIalpha and PIPKIgamma had higher kinase activity than PIPKIbeta. These results suggest that PIPKIalpha and PIPKIgamma are the critical priming factors for exocytosis; it also suggests that the levels of phosphatidylinositol phosphate kinase activity in producing phosphatidylinositol 4,5-bisphosphate specify the function of PIPKI isoforms in priming.
Wang
Li
L
Division of Cellular and Molecular Biology, Toronto Western Research Institute, University Health Network and Department of Physiology, University of Toronto, 399 Bathurst Street, Toronto, Ontario M5T 2S8, Canada.
Li
Gang
G
Sugita
Shuzo
S
D000595:Amino Acid Sequence; D000818:Animals; D005089:Exocytosis / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D016716:PC12 Cells; D010770:Phosphotransferases / Q000235:genetics / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D010770:Phosphotransferases