Phosphorylation of the activation loop of γ p21-activated kinase (γ-Pak) and related kinases (MSTs) in normal and stressed neutrophils

Neutrophils stimulated with a variety of chemoattractants exhibit a rapid activation of two p21-activated kinases (Paks) with molecular masses of similar to 63 and 69 kDa (gamma- and alpha -Pak). A number of in vitro studies suggest that modification of Thr(402) in the activation loop (AL) of gamma -Pak can play a critical role in the regulation of this kinase under certain circumstances. A phosphospecific Ab was generated to this region of Pak (pPak(AL)Ab). This Ab reacted with activated gamma- and alpha -Pak from fMLP-stimulated neutrophils that contain the sequence KRXT(P)XXGTP in their ALs. The rapid but transient activation of Paks in normal stimulated neutrophils coincided with phosphorylation and dephosphorylation at the ALs of these enzymes. In contrast, stressed cells exhibited a prolonged phosphorylation at Thr(402) in both intact gamma -Pak and a proteolytic fragment of this kinase. The pPak(AL)Ab also reacted with the mammalian sterile twenty-like kinases (MSTs) (members of the Pak family) in osmotically stressed neutrophils and neutrophils treated with certain apoptotic agents (i.e., tumor promoters that inhibit type 1 and 2A protein phosphatases) but not in normal fMLP-stimulated cells. Thus, our results indicate that the AL of gamma -Pak undergoes transient phosphorylation during normal neutrophil stimulation and chronic phosphorylation in stressed cells. In addition, we demonstrate that a number of MSTs are present in neutrophils and also undergo phosphorylation during stressful circumstances.