Protein kinases and phosphatases play key roles in integrating signals from various insulin secretagogues. In this study, we show that the activities of the cAMP-dependent protein kinase (PKA) and the calcium/calmodulin-dependent phosphatase, PP-2B are coordinated resulting in the regulation of insulin secretion. Transient inhibition of PP-2B, using the immunosuppressant FK506, increased forskolin stimulated insulin secretion by 2.5-fold +/- 0.3 (n = 6) in rat islets and RINm5F cells. Surprisingly, forskolin treatment resulted in the dephosphorylation of the vesicle-associated protein synapsin 1 and increased PP-2B activity by 2.98 +/- 0.97-fold (n = 4). One potential explanation for the observed coordination of PKA and PP-2B activity is their colocalization through a mutual anchoring protein, AKAP79/150. Accordingly, RINm5F cells expressing AKAP79 exhibited decreased insulin secretion, reduced PP-2B activity and were insensitive to FK506. This suggests that AKAP targeting of PKA and PP-2B maintains a signal transduction complex that may regulate reversible phosphorylation events involved in insulin secretion.