Antonio Casamayor

3-Phosphoinositide-dependent protein kinase-1 (PDK1): structural and functional homology with the Drosophila DSTPK61 kinase

BACKGROUNDnThe activation of protein kinase B (PKB, also known as c-Akt) is stimulated by insulin or growth factors and results from its phosphorylation at Thr308 and Ser473. We recently identified a protein kinase, termed PDK1, that phosphorylates PKB at Thr308 only in the presence of lipid vesicles containing phosphatidylinositol 3,4,5-trisphosphate (Ptdlns(3,4,5)P3) or phosphatidylinositol 3,4-bisphosphate (Ptdlns(3,4)P2).nnnRESULTSnWe have cloned and sequenced human PDK1. The 556-residue monomeric enzyme comprises a catalytic domain that is most similar to the PKA, PKB and PKC subfamily of protein kinases and a carboxy-terminal pleckstrin homology (PH) domain. The PDK1 gene is located on human chromosome 16p13.3 and is expressed ubiquitously in human tissues. Human PDK1 is homologous to the Drosophila protein kinase DSTPK61, which has been implicated in the regulation of sex differentiation, oogenesis and spermatogenesis. Expressed PDK1 and DSTPK61 phosphorylated Thr308 of PKB alpha only in the presence of Ptdlns(3,4,5)P3 or Ptdlns(3,4)P2. Overexpression of PDK1 in 293 cells activated PKB alpha and potentiated the IGF1-induced phosphorylation of PKB alpha at Thr308. Experiments in which the PH domains of either PDK1 or PKB alpha were deleted indicated that the binding of Ptdlns(3,4,5)P3 or Ptdlns(3,4)P2 to PKB alpha is required for phosphorylation and activation by PDK1. IGF1 stimulation of 293 cells did not affect the activity or phosphorylation of PDK1.nnnCONCLUSIONSnPDK1 is likely to mediate the activation of PKB by insulin or growth factors. DSTPK61 is a Drosophila homologue of PDK1. The effect of Ptdlns(3,4,5)P3/Ptdlns(3,4)P2 in the activation of PKB alpha is at least partly substrate directed.

PDK1 acquires PDK2 activity in the presence of a synthetic peptide derived from the carboxyl terminus of PRK2

BACKGROUNDnProtein kinase B (PKB) is activated by phosphorylation of Thr308 and of Ser473. Thr308 is phosphorylated by the 3-phosphoinositide-dependent protein kinase-1 (PDK1) but the identity of the kinase that phosphorylates Ser473 (provisionally termed PDK2) is unknown.nnnRESULTSnThe kinase domain of PDK1 interacts with a region of protein kinase C-related kinase-2 (PRK2), termed the PDK1-interacting fragment (PIF). PIF is situated carboxy-terminal to the kinase domain of PRK2, and contains a consensus motif for phosphorylation by PDK2 similar to that found in PKBalpha, except that the residue equivalent to Ser473 is aspartic acid. Mutation of any of the conserved residues in the PDK2 motif of PIF prevented interaction of PIF with PDK1. Remarkably, interaction of PDK1 with PIF, or with a synthetic peptide encompassing the PDK2 consensus sequence of PIF, converted PDK1 from an enzyme that could phosphorylate only Thr308 of PKBalpha to one that phosphorylates both Thr308 and Ser473 of PKBalpha in a manner dependent on phosphatidylinositol (3,4,5) trisphosphate (PtdIns(3,4,5)P3). Furthermore, the interaction of PIF with PDK1 converted the PDK1 from a form that is not directly activated by PtdIns(3,4,5)P3 to a form that is activated threefold by PtdIns(3,4,5)P3. We have partially purified a kinase from brain extract that phosphorylates Ser473 of PKBalpha in a PtdIns(3,4,5)P3-dependent manner and that is immunoprecipitated with PDK1 antibodies.nnnCONCLUSIONSnPDK1 and PDK2 might be the same enzyme, the substrate specificity and activity of PDK1 being regulated through its interaction with another protein(s). PRK2 is a probable substrate for PDK1.

Identification of a pocket in the PDK1 kinase domain that interacts with PIF and the C‐terminal residues of PKA

The 3‐phosphoinositide‐dependent protein kinase‐1 (PDK1) phosphorylates and activates a number of protein kinases of the AGC subfamily. The kinase domain of PDK1 interacts with a region of protein kinase C‐related kinase‐2 (PRK2), termed the PDK1‐interacting fragment (PIF), through a hydrophobic motif. Here we identify a hydrophobic pocket in the small lobe of the PDK1 kinase domain, separate from the ATP‐ and substrate‐binding sites, that interacts with PIF. Mutation of residues predicted to form part of this hydrophobic pocket either abolished or significantly diminished the affinity of PDK1 for PIF. PIF increased the rate at which PDK1 phosphorylated a synthetic dodecapeptide (T308tide), corresponding to the sequences surrounding the PDK1 phosphorylation site of PKB. This peptide is a poor substrate for PDK1, but a peptide comprising T308tide fused to the PDK1‐binding motif of PIF was a vastly superior substrate for PDK1. Our results suggest that the PIF‐binding pocket on the kinase domain of PDK1 acts as a ‘docking site’, enabling it to interact with and enhance the phosphorylation of its substrates.

Functional counterparts of mammalian protein kinases PDK1 and SGK in budding yeast

BACKGROUNDnIn animal cells, recruitment of phosphatidylinositol 3-kinase by growth factor receptors generates 3-phosphoinositides, which stimulate 3-phosphoinositide-dependent protein kinase-1 (PDK1). Activated PDK1 then phosphorylates and activates downstream protein kinases, including protein kinase B (PKB)/c-Akt, p70 S6 kinase, PKC isoforms, and serum- and glucocorticoid-inducible kinase (SGK), thereby eliciting physiological responses.nnnRESULTSnWe found that two previously uncharacterised genes of Saccharomyces cerevisiae, which we term PKH1 and PKH2, encode protein kinases with catalytic domains closely resembling those of human and Drosophila PDK1. Both Pkh1 and Pkh2 were essential for cell viability. Expression of human PDK1 in otherwise inviable pkh1Delta pkh2Delta cells permitted growth. In addition, the yeast YPK1 and YKR2 genes were found to encode protein kinases each with a catalytic domain closely resembling that of SGK; both Ypk1 and Ykr2 were also essential for viability. Otherwise inviable ypk1Delta ykr2Delta cells were fully rescued by expression of rat SGK, but not mouse PKB or rat p70 S6 kinase. Purified Pkh1 activated mammalian SGK and PKBalpha in vitro by phosphorylating the same residue as PDK1. Pkh1 activated purified Ypk1 by phosphorylating the equivalent residue (Thr504) and was required for maximal Ypk1 phosphorylation in vivo. Unlike PKB, activation of Ypk1 and SGK by Pkh1 did not require phosphatidylinositol 3,4,5-trisphosphate, consistent with the absence of pleckstrin homology domains in these proteins. The phosphorylation consensus sequence for Ypk1 was similar to that for PKBalpha and SGK.nnnCONCLUSIONSnPkh1 and Pkh2 function similarly to PDK1, and Ypk1 and Ykr2 to SGK. As in animal cells, these two groups of yeast kinases constitute two tiers of a signalling cascade required for yeast cell growth.

A 3-phosphoinositide-dependent protein kinase-1 (PDK1) docking site is required for the phosphorylation of protein kinase Czeta (PKCzeta ) and PKC-related kinase 2 by PDK1.

Members of the AGC subfamily of protein kinases including protein kinase B, p70 S6 kinase, and protein kinase C (PKC) isoforms are activated and/or stabilized by phosphorylation of two residues, one that resides in the T-loop of the kinase domain and the other that is located C-terminal to the kinase domain in a region known as the hydrophobic motif. Atypical PKC isoforms, such as PKCζ, and the PKC-related kinases, like PRK2, are also activated by phosphorylation of their T-loop site but, instead of possessing a phosphorylatable Ser/Thr in their hydrophobic motif, contain an acidic residue. The 3-phosphoinositide-dependent protein kinase (PDK1) activates many members of the AGC subfamily of kinases in vitro, including PKCζ and PRK2 by phosphorylating the T-loop residue. In the present study we demonstrate that the hydrophobic motifs of PKCζ and PKCι, as well as PRK1 and PRK2, interact with the kinase domain of PDK1. Mutation of the conserved residues of the hydrophobic motif of full-length PKCζ, full-length PRK2, or PRK2 lacking its N-terminal regulatory domain abolishes or significantly reduces the ability of these kinases to interact with PDK1 and to become phosphorylated at their T-loop sites in vivo. Furthermore, overexpression of the hydrophobic motif of PRK2 in cells prevents the T-loop phosphorylation and thus inhibits the activation of PRK2 and PKCζ. These findings indicate that the hydrophobic motif of PRK2 and PKCζ acts as a “docking site” enabling the recruitment of PDK1 to these substrates. This is essential for their phosphorylation by PDK1 in cells.

Characterisation of a plant 3-phosphoinositide-dependent protein kinase-1 homologue which contains a pleckstrin homology domain

A plant homologue of mammalian 3‐phosphoinositide‐dependent protein kinase‐1 (PDK1) has been identified in Arabidopsis and rice which displays 40% overall identity with human 3‐phosphoinositide‐dependent protein kinase‐1. Like the mammalian 3‐phosphoinositide‐dependent protein kinase‐1, Arabidopsis 3‐phosphoinositide‐dependent protein kinase‐1 and rice 3‐phosphoinositide‐dependent protein kinase‐1 possess a kinase domain at N‐termini and a pleckstrin homology domain at their C‐termini. Arabidopsis 3‐phosphoinositide‐dependent protein kinase‐1 can rescue lethality in Saccharomyces cerevisiae caused by disruption of the genes encoding yeast 3‐phosphoinositide‐dependent protein kinase‐1 homologues. Arabidopsis 3‐phosphoinositide‐dependent protein kinase‐1 interacts via its pleckstrin homology domain with phosphatidic acid, PtdIns3P, PtdIns(3,4,5)P3 and PtdIns(3,4)P2 and to a lesser extent with PtdIns(4,5)P2 and PtdIns4P. Arabidopsis 3‐phosphoinositide‐dependent protein kinase‐1 is able to activate human protein kinase Bα (PKB/AKT) in the presence of PtdIns(3,4,5)P3. Arabidopsis 3‐phosphoinositide‐dependent protein kinase‐1 is only the second plant protein reported to possess a pleckstrin homology domain and the first plant protein shown to bind 3‐phosphoinositides.