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

Neutrophils stimulated with a variety of chemoattractants exhibit a rapid a ctivation of two p21-activated kinases (Paks) with molecular masses of simi lar 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 certai n 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 neutrophi ls 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 k inase. The pPak(AL)Ab also reacted with the mammalian sterile twenty-like k inases (MSTs) (members of the Pak family) in osmotically stressed neutrophi ls and neutrophils treated with certain apoptotic agents (i.e., tumor promo ters that inhibit type 1 and 2A protein phosphatases) but not in normal fML P-stimulated cells. Thus, our results indicate that the AL of gamma -Pak un dergoes 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 phosphorylati on during stressful circumstances.