AKTide-2T

Heat shock protein 27 controls apoptosis by regulating Akt activation.[Pubmed:12740362]

J Biol Chem. 2003 Jul 25;278(30):27828-35.

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.

Peptide and protein library screening defines optimal substrate motifs for AKT/PKB.[Pubmed:10945990]

J Biol Chem. 2000 Nov 17;275(46):36108-15.

AKT was originally identified as a proto-oncogene with a pleckstrin homology and Ser/Thr protein kinase domains. Recent studies revealed that AKT regulates a variety of cellular functions including cell survival, cell growth, cell differentiation, cell cycle progression, transcription, translation, and cellular metabolism. To clarify the substrate specificity of AKT, we have used an oriented peptide library approach to determine optimal amino acids at positions N-terminal and C-terminal to the site of phosphorylation. The predicted optimal peptide substrate (Arg-Lys-Arg-Xaa-Arg-Thr-Tyr-Ser*-Phe-Gly where Ser* is the phosphorylation site) has similarities to but is distinct from optimal substrates that we previously defined for related basophilic protein kinases such as protein kinase A, Ser/Arg-rich kinases, and protein kinase C family members. The positions most important for high V(max)/K(m) ratio were Arg-3>Arg-5>Arg-7. The substrate specificity of AKT was further investigated by screening a lambdaGEX phage HeLa cell cDNA expression library. All of the substrates identified by this procedure contained Arg-Xaa-Arg-Xaa-Xaa-(Ser/Thr) motifs and were in close agreement with the motif identified by peptide library screening. The results of this study should help in prediction of likely AKT substrates from primary sequences.