Mast cells permeabilized by streptolysin O undergo exocytosis when stimulated with Ca2+ and guanosine 5′-[γ-thio]triphosphate but become progressively refractory to this stimulus if it is delayed. This run-down of responsiveness occurs over a period of 20–30min, during which the cells leak soluble and tethered proteins. We show here that withdrawal of ATP during the process of run-down is strongly inhibitory but that as little as 25μM ATP can extend responsiveness significantly; this effect is maximal at 50μM. When phosphatidylinositol transfer proteins (PITPs) are provided to cells at the time of permeabilization, run-down is retarded. We conclude that in the presence of ATP they convey substrates for phosphorylation that are essential for exocytosis and thus interact with the regulatory machinery. Furthermore, we show that PITPα and PITPβ have additive effects in this mechanism, suggesting that they are not functionally redundant. Alternatively, secretion from run-down cells can be inhibited by the aminoglycoside antibiotic neomycin, which is understood to bind to phosphoinositide headgroups, and by a PH (pleckstrin homology) domain polypeptide that binds phosphoinositides. The apparent displacement of neomycin by exogenous PITPs suggests that these proteins screen essential lipids. Secretion from run-down cells is also inhibited by 1-O-hexadecyl-2-O-methyl-rac-glycerol (AMG-C16), an inhibitor of protein kinase C. The lack of synergy between neomycin and AMG-C16 suggests that protein kinase C independently provides a second essential component through protein phosphorylation and that there are two independent phosphorylation pathways necessary for secretion competence.
Skip Nav Destination
Article navigation
Research Article|
May 08 2001
Phosphatidylinositol transfer proteins and protein kinase C make separate but non-interacting contributions to the phosphorylation state necessary for secretory competence in rat mast cells
Jef A. PINXTEREN;
Jef A. PINXTEREN
1
∗Secretory Mechanisms Group, Department of Physiology, University College London, London WC1E 6JJ, U.K.
1To whom correspondence should be addressed. Present address: Laboratories for Paediatrics and Immunology, Department of Medicine, University of Antwerp, T4.30, Universiteitsplein 1, 2610 Wilrijk, Belgium (e-mail jef.pinxteren@ua.ac.be).
Search for other works by this author on:
Bastien D. GOMPERTS;
Bastien D. GOMPERTS
∗Secretory Mechanisms Group, Department of Physiology, University College London, London WC1E 6JJ, U.K.
Search for other works by this author on:
Danise ROGERS;
Danise ROGERS
†Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL 35294-0005, U.S.A.
Search for other works by this author on:
Scott E. PHILLIPS;
Scott E. PHILLIPS
†Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL 35294-0005, U.S.A.
Search for other works by this author on:
Peter E. R. TATHAM;
Peter E. R. TATHAM
∗Secretory Mechanisms Group, Department of Physiology, University College London, London WC1E 6JJ, U.K.
Search for other works by this author on:
Geraint M. H. THOMAS
Geraint M. H. THOMAS
∗Secretory Mechanisms Group, Department of Physiology, University College London, London WC1E 6JJ, U.K.
Search for other works by this author on:
Publisher: Portland Press Ltd
Received:
January 02 2001
Revision Received:
February 15 2001
Accepted:
March 15 2001
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2001
2001
Biochem J (2001) 356 (1): 287–296.
Article history
Received:
January 02 2001
Revision Received:
February 15 2001
Accepted:
March 15 2001
Citation
Jef A. PINXTEREN, Bastien D. GOMPERTS, Danise ROGERS, Scott E. PHILLIPS, Peter E. R. TATHAM, Geraint M. H. THOMAS; Phosphatidylinositol transfer proteins and protein kinase C make separate but non-interacting contributions to the phosphorylation state necessary for secretory competence in rat mast cells. Biochem J 15 May 2001; 356 (1): 287–296. doi: https://doi.org/10.1042/bj3560287
Download citation file:
Sign in
Don't already have an account? Register
Sign in to your personal account
You could not be signed in. Please check your email address / username and password and try again.
Captcha Validation Error. Please try again.