Scientific Report 2005

Molecular and Experimental Medicine

Division Of Biochemistry

Mechanisms of Regulated Secretion

S.D. Catz, J.L. Johnson, B.A. Ellis, J.M. Ruedi, D. Noack, S. Pacquelet, B. Young

A General Role for Rab27 And JFC1 in Secretory Cells

The traffic of secretory vesicles to the plasma membrane in eukaryotic cells is essential for normal cell function. The specificity of vesicular transport relies on Rab GTPases that act as membrane organizers and on their specific effectors that transduce many steps in cell trafficking. Rab27a, the only Rab known to associate directly with a disease in humans (Griscelli syndrome), plays a central role in regulated secretion. We are studying the mechanisms involved in vesicle exocytosis, in particular, the function of Rab27a effectors in this process.

Recently, we identified JFC1/Slp1, a member of the synaptotagmin-like protein family of effectors characterized by the presence of a Rab27-binding motif in its amino terminus and by C2 domains capable of binding phosphatidylinositol-3,4,5-trisphosphate in its carboxy terminus. Using a combination of functional analysis and confocal microscopy, we discovered that JFC1 and Rab27a regulate the androgen-dependent secretion of prostatic-specific acid phosphatase and prostate-specific antigen in human prostate carcinoma cells in a process that involves the phosphatidylinositol-3´-kinase pathway.

In other studies, we have made progress in characterizing the secretory machinery used by the many secretory organelles present in human neutrophils. Neutrophils have at least 4 distinct secretory organelles. During activation and phagocytosis, neutrophils can differentially secret the contents of the organelles into the acceptor compartment and/or integrate vesicular proteins (e.g., cytochrome b₅₅₈) into acceptor membranes. The secretion of these organelles is thought to be hierarchical. We aim to identify the secretory machineries that control the differential secretory behavior of these organelles.

Both the prostate and the neutrophil secretion studies will be complemented by using the ashen (Rab27a^ash) mouse model.

IL-1 Receptor–Associated Kinase-4 and NADPH Oxidase

For more than 20 years, it has been known that lipopolysaccharide can elicit neutrophil priming for the production of superoxide anion in response to formylated peptides. This phenomenon has been explained in part by the ability of lipopolysaccharide to upregulate the NADPH oxidase assembly. However, it is still unclear how the signaling downstream activation of Toll-like receptor 4 is involved in the upregulation of the NADPH oxidase. Recently, we discovered that IL-1 receptor–associated kinase-4, a kinase involved in the signaling downstream activation of Toll-like receptors, phosphorylates the NADPH oxidase cytosolic factor p47^phox. Using mass spectrometric analysis, we identified a threonine-rich domain in p47^phox that is the target of phosphorylation by the kinase. We are analyzing the physiologic importance of these findings.

Publications

Johnson, J.L., Pacquelet, S., Lane, W.S., Eam, B., Catz, S.D. Akt regulates the subcellular localization of the Rab27a-binding protein JFC1 by phosphorylation. Traffic 6:667, 2005.