p21-activated kinase 2 in neutrophils can be regulated by phosphorylation at multiple sites and by a variety of protein phosphatases

J Immunol. 2003 Oct 1;171(7):3785-93. doi: 10.4049/jimmunol.171.7.3785.

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 (Thr(402)) and inhibitory domain (Ser(141)) is known to increase the activity of Pak in vitro, whereas phosphorylation within the Nck (Ser(20)) and Pak-interacting guanine nucleotide exchange factor (Ser(192) and Ser(197)) 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 (< or =15 s) phosphorylation at Ser(20), Ser(192/197), and Thr(402) in neutrophils stimulated with fMLP. Phosphorylation at Ser(192/197) and Thr(402) were highly transient events, whereas that at Ser(20) was more persistent. In contrast, Pak 2 was constitutively phosphorylated at Ser(141) 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 Thr(402) 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 Mg(2+)/Mn(2+)-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.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cell Fractionation
  • Enzyme Activation / physiology
  • Enzyme Inhibitors / pharmacology
  • Glutathione Transferase / genetics
  • Glutathione Transferase / metabolism
  • Guinea Pigs
  • Humans
  • Mice
  • Molecular Sequence Data
  • N-Formylmethionine Leucyl-Phenylalanine / pharmacology
  • Neutrophil Activation / physiology
  • Neutrophils / enzymology*
  • Neutrophils / metabolism
  • Neutrophils / physiology
  • Oncogene Protein p21(ras) / physiology*
  • Phosphoprotein Phosphatases / antagonists & inhibitors
  • Phosphoprotein Phosphatases / physiology*
  • Phosphorylation / drug effects
  • Protein Phosphatase 1
  • Protein-Serine-Threonine Kinases / genetics
  • Protein-Serine-Threonine Kinases / metabolism*
  • Recombinant Fusion Proteins / metabolism
  • Serine / metabolism
  • Threonine / metabolism
  • p21-Activated Kinases

Substances

  • Enzyme Inhibitors
  • Recombinant Fusion Proteins
  • Threonine
  • Serine
  • N-Formylmethionine Leucyl-Phenylalanine
  • Glutathione Transferase
  • PAK2 protein, human
  • PAK3 protein, human
  • Pak2 protein, mouse
  • Pak3 protein, mouse
  • Protein-Serine-Threonine Kinases
  • p21-Activated Kinases
  • Phosphoprotein Phosphatases
  • Protein Phosphatase 1
  • Oncogene Protein p21(ras)