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. Author manuscript; available in PMC: 2011 Mar 11.
Published in final edited form as: Cell Metab. 2011 Mar 2;13(3):308–319. doi: 10.1016/j.cmet.2011.02.002

Figure 5. PKA mediated phosphorylation of snapin maps to serine 50. Snapin S50 phosphorylation increases interaction with secretory vesicle-associated proteins SNAP-25, EPAC2 and collectrin. Snapin interaction with VAMP2 is glucose dependent. E4 stimulated SNAP-25 interaction with EPAC2 is not PKA mediated. Overexpression in islets of snapin S50D potentiates GSIS.

Figure 5

A) Transiently transfected INS1 832/13 cells expressing C-terminal FLAG tagged WT, S50A or S50D snapin were treated with PBS (vehicle) E4 (10 nM), myr-PKI (10 nM), treated E4+myr-PKI, or transfected with Pkac followed by IP for FLAG or SNAP-25 and IB for phosphoserine or interacting proteins. Snapin serine phosphorylation occurs in WT snapin and not in S50A and S50D mutants by E4 and Pkac action. E4 effect is inhibited by myr-PKI. Snapin interaction with SNAP-25, collectrin or EPAC2 occurs only with phosphorylated WT snapin (by E4 or Pkac) or with snapin S50D as does SNAP-25 interaction with collectrin. SNAP-25 interaction with EPAC2 occurs with E4 in a PKA-independent manner and not inhibited by myr-PKI.

B) Transiently transfected INS1 832/13 cells expressing C-terminal FLAG tagged WT, S50A or S50D snapin cultured in low (3 mM) or high (10 mM) glucose and co-IP/IB as in A. Snapin S50D mutant binds SNAP-25, collectrin and EPAC2 in both low and high glucose. Snapin interaction with VAMP2 occurs at elevated glucose levels only. SNAP-25- VAMP2 interaction occurs only with snapin S50D and at elevated glucose levels.

C) Isolated C57Bl/6 mouse islets cultured in low (3 mM) or high (10 mM) glucose and treated with PBS, E4 (10 nM), myr-PKI (10 nM) and E4+myr-PKI followed by co-IP/IB as in A. Snapin serine phosphorylation is stimulated by E4 in a PKA dependent manner and increases snapin interaction with SNAP-25, collectrin and EPAC2 independently of glucose levels. Snapin-VAMP2 interaction is stimulated by E4 in PKA dependent manner only with high glucose. E4 stimulates SNAP25-EPAC2 interaction in a glucose- and PKA-independent manner. SNAP-25-VAMP2 interaction is stimulated by E4 in a glucose and PKA-dependent manner. PKA activity is verified by phosphorylation of CREB at serine 133 (p-CREB).

D) Control and Δ-prkar1a islets cultured in low (3 mM) or high glucose (10 mM). IP for snapin or SNAP-25 and IB for phosphoserine and interacting proteins. Snapin phosphorylation and snapin interaction with SNAP-25, collectrin and EPAC2 are increased in Δ-prkar1a islets independently of glucose levels. Snapin-VAMP2 and SNAP-25-VAMP2 interactions are stimulated by high glucose only.

E) Perifusion studies of C57Bl/6 mouse islets in low (3 mM) followed by high (10 mM) glucose concentrations. Islets were treated with either PBS (inverted triangle), E4 (10 nM) (upright triangle) during perifusion, or had been transduced with control (circle) or snapin S50D (square) expressing adenovirus. Table below curve summarizes area under the curves for first and second phase insulin secretion (*P<0.05 vs vehicle).