14-337 Sigma-AldrichMAPKAP Kinase 2 Protein, active, 10 µg

Activation of the serine-threonine kinase Akt by cytokines, chemokines, and bacterial products delays constitutive neutrophil apoptosis, resulting in a prolonged inflammatory response. We showed previously that Akt exists in a signaling complex with p38 MAPK, MAPK-activated protein kinase-2 (MAPKAPK-2), and heat shock protein-27 (Hsp27); and Hsp27 dissociates from the complex upon neutrophil activation. To better understand the regulation of this signaling module, the hypothesis that Akt phosphorylation of Hsp27 regulates its interaction with Akt was tested. The present study shows that Akt phosphorylated Hsp27 on Ser-82 in vitro and in intact cells, and phosphorylation of Hsp27 resulted in its dissociation from Akt. Additionally, the interaction between Hsp27 and Akt was necessary for activation of Akt in intact neutrophils. Constitutive neutrophil apoptosis was accelerated by sequestration of Hsp27 from Akt, and this enhanced rate of apoptosis was reversed by introduction of constitutively active recombinant Akt. Our results define a new mechanism by which Hsp27 regulates apoptosis, through control of Akt activity.

A novel protein kinase, which was only active when phosphorylated by the mitogen-activated protein kinase (MAP kinase), has been purified 85,000-fold to homogeneity from rabbit skeletal muscle. This MAP kinase activated protein kinase, termed MAPKAP kinase-2, was distinguished from S6 kinase-II (MAPKAP kinase-1) by its response to inhibitors, lack of phosphorylation of S6 peptides and amino acid sequence. MAPKAP kinase-2 phosphorylated glycogen synthase at Ser7 and the equivalent serine (*) in the peptide KKPLNRTLS*VASLPGLamide whose sequence is similar to the N terminus of glycogen synthase. MAPKAP kinase-2 was resolved into two monomeric species of apparent molecular mass 60 and 53 kDa that had similar specific activities and substrate specificities. Peptide sequences of the 60 and 53 kDa species were identical, indicating that they are either closely related isoforms or derived from the same gene. MAP kinase activated the 60 and 53 kDa forms of MAPKAP kinase-2 by phosphorylating the first threonine residue in the sequence VPQTPLHTSR. Furthermore, Mono Q chromatography of extracts from rat phaeochromocytoma and skeletal muscle demonstrated that two MAP kinase isoforms (p42mapk and p44mapk) were the only enzymes in these cells that were capable of reactivating MAPKAP kinase-2. These results indicate that MAP kinase activates at least two distinct protein kinases, suggesting that it represents a point at which the growth factor-stimulated protein kinase cascade bifurcates.