Protein kinase-ζ interacts with munc18c: role in GLUT4 trafficking

Abstract

Aims/hypothesis: Insulin-stimulated glucose transport requires a signalling cascade through kinases protein kinase (PK) Cζ/λ and PKB that leads to movement of GLUT4 vesicles to the plasma membrane. The aim of this study was to identify missing links between the upstream insulin-regulated kinases and the GLUT4 vesicle trafficking system. Materials and methods: A yeast twohybrid screen was conducted, using as bait full-length mouse munc18c, a protein known to be part of the GLUT4 vesicle trafficking machinery. Results: The yeast twohybrid screen identified PKCζ as a novel interactor with munc18c. Glutathione S transferase (GST) pull-downs with GST-tagged munc18c constructs confirmed the interaction, mapped a key region of munc18c that binds PKCζ to residues 295-338 and showed that the N-terminal region of PKCζ was required for the interaction. Endogenous munc18c was shown to associate with endogenous PKCζ in vivo in various cell types. Importantly, insulin stimulation increased the association by approximately three-fold. Moreover, disruption of PKCζ binding to munc18c by deletion of residues 295-338 of munc18c or deletion of the N-terminal region of PKCζ markedly inhibited the ability of insulin to stimulate glucose uptake or GLUT4 translocation. Conclusions/interpretation: We have identified a physiological interaction between munc18c and PKCζ that is insulin-regulated. This establishes a link between a kinase (PKCζ) involved in the insulin signalling cascade and a known component of the GLUT4 vesicle trafficking pathway (munc18c). The results indicate that PKCζ regulates munc18c and suggest a model whereby insulin triggers the docking of PKCζ to munc18c, resulting in enhanced GLUT4 translocation to the plasma membrane.

Munc18c Interaction with Syntaxin 4 Monomers and SNARE Complex Intermediates in GLUT4 Vesicle Trafficking

“…Several lines of evidence suggest that insulin ultimately reduces Munc18c-syntaxin 4 complexation through the activity of phosphatidylinositol 3-kinase and protein kinase C (10,11). However, it appears that dissociation of Munc18c from syntaxin 4 may only be required at a site downstream of SNARE complex formation and committed vesicle docking.…”

“…The delivery of GLUT4 to the cell surface occurs by distinct signaling processes: 1) GLUT4 vesicle recruitment to the plasma membrane, followed by 2) phosphatidylinositol 3-kinase-dependent vesicle docking and fusion mediated by syntaxin 4-containing SNARE complexes (9,10). Based on overexpression studies, Munc18c was initially proposed to function as a negative regulator of GLUT4 vesicle translocation in 3T3L1 adipocytes (5,6).…”

Ins (endocytosis) and outs (exocytosis) of GLUT4 trafficking

“…However another study, found that homozygous Munc18c knockout mice were viable and had increased insulin sensitivity consistent with Munc18c actin as a fusogenic inhibitor [63]. Insulin has also been observed to induce the association of PKC and Munc18c concomitant with a dissociation of the syntaxin4/Munc18c complex [50]. More over, insulin triggers PKC forms a complex with 80K-H and munc18c to promote VAMP2 binding to syntaxin4 that enhanced the GLUT4 fusion with the plasma membrane.…”

“…Several syntaxin 4 binding partners have been identified including one member of the Munc18 family (Munc18c), Synip and Tomosyn [47,50,51]. Although the potential role of Tomosyn has not been well studied, the evidence supporting a critical role for either Munc18c and/or Synip has remained controversial.…”

The Role of Phosphoinositide 3-Kinase C2α in Insulin Signaling

“…6). In this respect, recent evidence suggests that Akt might be the PI3K target necessary for tethering and docking of GSVs (33), whereas protein kinase C and Munc18c (41,70) might be the PI3K targets involved in fusion. A critical role for Akt in fusion has also been proposed (66), indicating that work still needs to be done to precisely define the PI3K-dependent players in the different steps.…”