p21-activated kinase 1 activity is required for histone H3 Ser10 phosphorylation and chromatin condensation in mouse oocyte meiosis

p21-activated kinase 1 (Pak1) is essential for a variety of cellular events, including gene transcription, cytoskeletal organisation, cell proliferation and apoptosis. Pak1 is activated upon autophosphorylation on many amino residues; in particular, phosphorylation on Thr423 maintains maximal Pak1 activation. In the present study we investigated the protein expression, subcellular localisation and function of Pak1 phosphorylated on Thr423 (pPak1Thr423) in mouse oocytes. pPak1Thr423 was detected upon meiotic resumption and localised on the condensing chromatin. Thr423 phosphorylation was markedly suppressed by the Pak1 ATP-competitive inhibitor PF-3758309, but not by the allosteric inhibitors IPA-3 (2.5 μM and 10 μM) (1, 1′-dithiobis-2-naphthalenol) and TAT-PAK18 (10 μM), which prevent the binding of Pak1 to its upstream activators GTPase Cdc42/Rac and Pak-interacting exchange factor (PIX), respectively, implying that Pak1 activation may be independent of GTPase and PIX in oocyte meiosis. Inhibition of Pak1 activation concomitantly restrained histone H3 phosphorylation on Ser10 and consequently inhibited chromatin condensation; however, this phenotype was reversed by concomitant administration of the Pak1 activator FTY720. The changes in the pattern of expression of phosphorylated extracellular signal-regulated kinase 1/2 in response to PF-3758309 or FTY720 were the same as seen for pPak1Thr423. These results show that activated Pak1 regulates chromatin condensation by promoting H3 Ser10 phosphorylation in oocytes after the resumption of meiotic progression.

Protein kinase CK2 phosphorylates and activates p21-activated kinase 1

Activation of the p21-activated kinase 1 (PAK1) is achieved through a conformational change that converts an inactive PAK1 dimer to an active monomer. In this paper, we show that this change is necessary but not sufficient to activate PAK1 and that it is, rather, required for CK2-dependent PAK1S223 phosphorylation that converts a monomeric PAK1 into a catalytically active form. This phosphorylation appears to be essential for autophosphorylation at specific residues and overall activity of PAK1. A phosphomimetic mutation (S223E) bypasses the requirement for GTPases in PAK1 activation, whereas the constitutive activity of the PAK1 mutant (PAK1H83,86L), postulated to mimic GTPase-induced structural changes, is abolished by inhibition of S223 phosphorylation. Thus, S223 is likely accessible to CK2 upon conformational changes of PAK1 induced by GTPase-dependent and GTPase-independent stimuli, suggesting that S223 phosphorylation may play a key role in the final step of the PAK1 activation process. The physiological significance of this phosphorylation is reinforced by the observations that CK2 is responsible for epidermal growth factor–induced PAK1 activation and that inhibition of S223 phosphorylation abrogates PAK1-mediated malignant transformation of prostate epithelial cells. Taken together, these findings identify CK2 as an upstream activating kinase of PAK1, providing a novel mechanism for PAK1 activation.