Protein kinase C delta (PKCd) is a Ser/Thr kinase which regulates numerous cellular processes, including proliferation, differentiation, migration and apoptosis. Here, we demonstrate that PKCd undergoes in vitro autophosphorylation at three sites within its V3 region (S299, S302, S304), each of which is unique to this PKC isoform and evolutionarily conserved. We demonstrate that S299 and S304 can be phosphorylated in mammalian cells following phorbol ester stimulation and that S299-phosphorylated PKCd is localised to both the plasma and nuclear membranes. These data indicate that PKCd is phosphorylated upon activation and that phospho-S299 represents a useful marker of the activated enzyme.

The S6 kinases (S6Ks) have been linked to a number of cellular processes, including translation, insulin metabolism, cell survival, and RNA splicing. Signaling via the phosphotidylinositol 3-kinase and mammalian target of rapamycin (mTOR) pathways is critical in regulating the activity and subcellular localization of S6Ks. To date, nuclear functions of both S6K isoforms, S6K1 and S6K2, are not well understood. To better understand S6K nuclear roles, we employed affinity purification of S6Ks from nuclear preparations followed by mass spectrometry analysis for the identification of novel binding partners. In this study, we report that in contrast to S6K1, the S6K2 isoform specifically associates with a number of RNA-binding proteins, including heterogeneous ribonucleoproteins (hnRNPs). We focused on studying the mechanism and physiological relevance of the S6K2 interaction with hnRNP F/H. Interestingly, the S6K2-hnRNP F/H interaction was not affected by mitogenic stimulation, whereas mTOR binding to hnRNP F/H was induced by serum stimulation. In addition, we define a new role of hnRNP F in driving cell proliferation, which could be partially attenuated by rapamycin treatment. S6K2-driven cell proliferation, on the other hand, could be blocked by small interfering RNA-mediated down-regulation of hnRNP F. These results demonstrate that the specific interaction between mTOR and S6K2 with hnRNPs is implicated in the regulation of cell proliferation.Regulation of cellular processes by extracellular stimuli, such as growth factors and nutrients, is an essential process in cell size and cell cycle control. The mammalian target of rapamycin (mTOR) 2 is a key regulator of cellular signaling and is highly dependent on the presence of nutrients, as well as growth factor-stimulated signaling from the phosphotidylinositol 3-kinase (PI3K) pathway. Activation of PI3K by a number of growth factor receptors leads to increased levels of phosphatidylinositol 3,4,5-triphosphate (1), a secondary messenger that recruits downstream kinases, such as 3-phosphoinositide-dependent kinase 1 (PDK1), to the plasma membrane (2). Membrane-associated PDK1 in turn phosphorylates and activates Akt at the T loop (3, 4), whereas activated Akt phosphorylation of tuberous sclerosis complex 2 (TSC2) blocks its inhibitory role in mTOR signaling (5, 6). TSC2 forms a heterodimer with TSC1, which as a complex acts as a GTPase activator toward the small GTPase, Rheb (Ras homolog enriched in brain) (7,8). Rheb associates directly with the catalytic domain of mTOR and activates it by antagonizing FKBP38, the proposed endogenous inhibitor of mTOR (9). Activated mTOR with its interacting partner protein Raptor, as part of the mTOR complex I (mTORC1) is able to phosphorylate S6Ks (at Thr 389 in S6K1 and Thr 388 in S6K2) (10). This early phosphorylation event is required for subsequent phosphorylation by PDK1 of Thr 229 and Thr 228 in the activation loops of S6K1 and S6K2, respectively (11). Phosphorylation by both mTORC1 and PDK1 leads to S6K activation, resul...

The PRKs [protein kinase C-related kinases; also referred to as PKNs (protein kinase Ns)] are a kinase family important in diverse functions including migration and cytokinesis. In the present study, we have re-evaluated and compared the specificity of PKN1 and PKN3 and assessed the predictive value in substrates. We analysed the phosphorylation consensus motif of PKNs using a peptide library approach and demonstrate that both PKN1 and PKN3 phosphorylate serine residues in sequence contexts that have an arginine residue in position -3. In contrast, PKN1 and PKN3 do not tolerate arginine residues in position +1 and -1 respectively. To test the predictive value of this motif, site analysis was performed on the PKN substrate CLIP-170 (cytoplasmic linker protein of 170 kDa); a PKN target site was identified that conformed to the predicted pattern. Using a protein array, we identified 22 further substrates for PKN1, of which 20 were previously undescribed substrates. To evaluate further the recognition signature, the site on one of these hits, EGFR (epidermal growth factor receptor), was identified. This identified Thr⁶⁵⁴ in EGFR as the PKN1 phosphorylation site and this retains an arginine residue at the -3 position. Finally, the constitutive phosphorylation of EGFR on Thr⁶⁵⁴ is shown to be modulated by PKN in vivo.