p21-Activated Kinase 2 in Neutrophils Can Be Regulated by Phosphorylation at Multiple Sites and by a Variety of Protein Phosphatases

Abstract

The p21-activated kinase(Pak) 2 undergoes rapid autophosphorylation/activation in neutrophils stimulated with a variety of chemoattractants (e.g., fMLP). Phosphorylation within the activation loop (Thr402) and inhibitory domain (Ser141) is known to increase the activity of Pak in vitro, whereas phosphorylation within the Nck (Ser20) and Pak-interacting guanine nucleotide exchange factor (Ser192 and Ser197) binding sites blocks the interactions of Pak 2 with these proteins. A panel of phosphospecific Abs was used to investigate the phosphorylation of Pak 2 in neutrophils at these sites. Pak 2 underwent rapid (≤15 s) phosphorylation at Ser20, Ser192/197, and Thr402 in neutrophils stimulated with fMLP. Phosphorylation at Ser192/197 and Thr402 were highly transient events, whereas that at Ser20 was more persistent. In contrast, Pak 2 was constitutively phosphorylated at Ser141 in unstimulated neutrophils and phosphorylation at this site was less sensitive to cell stimulation than at other residues. Studies with selective inhibitors suggested that a variety of phosphatases might be involved in the rapid dephosphorylation of Pak 2 at Thr402 in stimulated neutrophils. This was consistent with biochemical studies which showed that the activation loop of GST-Pak 3, which is homologous to that in Pak 2, was a substrate for protein phosphatase 1, 2A, and a Mg2+/Mn2+-dependent phosphatase(s) which exhibited properties different from those of the conventional isoforms of protein phosphatase 2C. The data indicate that Pak 2 undergoes a complex pattern of phosphorylation in neutrophils and that dephosphorylation at certain sites may involve multiple protein phosphatases that exhibit distinct modes of regulation.