Partial purification and characterization of a wortmannin-sensitive and insulin-stimulated protein kinase that activates heart 6-phosphofructo-2-kinase

A wortmannin-sensitive and insulin-stimulated protein kinase (WISK), which phosphorylates and activates cardiac 6-phosphofructo-2-kinase (PFK-2), was partially purified from perfused rat hearts. Immunoblotting showed that WIS K was devoid of protein kinase B (PKB), serum- and glucocorticoid-regulated protein kinase and protein kinase C zeta (PKC zeta). Comparison of the inh ibition of WISK, PKC alpha and PKC zeta by different protein kinase inhibit ors suggested that WISK was not a member of the PKC family. In addition, WI SK contained no detectable phosphoinositide-dependent protein kinase-1 (PDK 1) activity. WISK phosphorylated recombinant heart PFK-2 in a time-dependen t manner to the extent of 0.4 mol of phosphate incorporated/mol of enzyme s ubunit, and increased the V-max of PFK-2 twofold, without affecting the K-m for fructose 6-phosphate, WISK phosphorylated Ser-466 to a greater extent than Ser-483 in recombinant heart PFK-2, and both sites were demonstrated t o be phosphorylated to the same extent by PKB, Gel filtration and in-gel ki nase analysis indicated that WISK was a monomer with a M-r of 56500. Treatm ent of WISK with protein phosphatase 2A (PP2A) catalytic subunits reversed the effect of insulin, suggesting the involvement of an upstream activating kinase, Indeed, PDK1 was able to partially reactivate the PP2A-treated WIS K and this reactivation was not enhanced by PtdIns(3,4,5)P-3-containing ves icles. Moreover, a single 57000-M-r band was labelled on incubation of the dephosphorylated WISK preparation with PDK1 and [gamma-P-32]ATP. These find ings provide evidence for the existence of a new protein kinase in the insu lin signalling pathway, probably downstream of PDK1.