A chimeric mechanism for polyvalent trans-phosphorylation of PKA by PDK1.
ABSTRACT: Phosphorylation on the activation loop of AGC kinases is typically mediated by PDK1. The precise mechanism for this in-trans phosphorylation is unknown; however, docking of a hydrophobic (HF) motif in the C-tail of the substrate kinase onto the N-lobe of PDK1 is likely an essential step. Using a peptide array of PKA to identify other PDK1-interacting sites, we discovered a second AGC-conserved motif in the C-tail that interacts with PDK1. Since this motif [FD(X)(1-2)Y/F] lies in the active site tether region and in PKA contributes to ATP binding, we call it the Adenosine binding (Ade) motif. The Ade motif is conserved as a PDK1-interacting site in Akt and PRK2, and we predict it will be a PDK1-interacting site for most AGC kinases. In PKA, the HF motif is only recognized when the turn motif Ser338 is phosphorylated, possibly serving as a phosphorylation "switch" that regulates how the Ade and HF motifs interact with PDK1. These results demonstrate that the extended AGC C-tail serves as a polyvalent element that trans-regulates PDK1 for catalysis. Modeling of the PKA C-tail onto PDK1 structure creates two chimeric sites; the ATP binding pocket, which is completed by the Ade motif, and the C-helix, which is positioned by the HF motif. Together, they demonstrate substrate-assisted catalysis involving two kinases that have co-evolved as symbiotic partners. The highly regulated turn motifs are the most variable part of the AGC C-tail. Elucidating the highly regulated cis and trans functions of the AGC tail is a significant future challenge.
Project description:3-phosphoinositide dependent protein kinase-1 (PDK1) plays a key role in regulating signalling pathways by activating AGC kinases such as PKB/Akt and S6K. Here we describe the 2.0 A crystal structure of the PDK1 kinase domain in complex with ATP. The structure defines the hydrophobic pocket termed the "PIF-pocket", which plays a key role in mediating the interaction and phosphorylation of certain substrates such as S6K1. Phosphorylation of S6K1 at its C-terminal PIF-pocket-interacting motif promotes the binding of S6K1 with PDK1. In the PDK1 structure, this pocket is occupied by a crystallographic contact with another molecule of PDK1. Interestingly, close to the PIF-pocket in PDK1, there is an ordered sulfate ion, interacting tightly with four surrounding side chains. The roles of these residues were investigated through a combination of site-directed mutagenesis and kinetic studies, the results of which confirm that this region of PDK1 represents a phosphate-dependent docking site. We discuss the possibility that an analogous phosphate-binding regulatory motif may participate in the activation of other AGC kinases. Furthermore, the structure of PDK1 provides a scaffold for the design of specific PDK1 inhibitors.
Project description:The catalytic activities of eukaryotic protein kinases (EPKs) are regulated by movement of the C-helix, movement of the N and C lobes upon ATP binding, and movement of the activation loop upon phosphorylation. Statistical analysis of the selective constraints associated with AGC kinase functional divergence reveals conserved interactions between these regulatory regions and three regions of the C-terminal tail (C-tail): the N-lobe tether (NLT), the active-site tether (AST), and the C-lobe tether (CLT). The NLT serves as a docking site for an upstream kinase PDK1 and, upon activation, positions the C-helix within the ATP binding pocket. The AST directly interacts with the ATP binding pocket, and the CLT interacts with the interlobe linker and the alphaC-beta4 loop, which appears to serve as a hinge for C-helix movement. The C-tail is a hallmark of AGC functional divergence inasmuch as most of the conserved core residues that distinguish AGC kinases from other EPKs are associated with the NLT, AST, or CLT. Moreover, several AGC catalytic core conserved residues that interact with the C-tail strikingly diverge from the canonical residues observed at corresponding positions in nearly all other EPKs, suggesting that the catalytic core may have coevolved with the C-tail in AGC kinases. These observations, along with the fact that the C-tail is needed for catalytic activity suggests that the C-tail is a cis-acting regulatory module that can also serve as a regulatory "handle," to which trans-acting cellular components can bind to modulate activity.
Project description:The members of the AGC kinase family frequently exhibit three conserved phosphorylation sites: the activation loop, the hydrophobic motif (HM), and the zipper (Z)/turn-motif (TM) phosphorylation site. 3-Phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylates the activation loop of numerous AGC kinases, including the protein kinase C-related protein kinases (PRKs). Here we studied the docking interaction between PDK1 and PRK2 and analyzed the mechanisms that regulate this interaction. In vivo labeling of recombinant PRK2 by (32)P(i) revealed phosphorylation at two sites, the activation loop and the Z/TM in the C-terminal extension. We provide evidence that phosphorylation of the Z/TM site of PRK2 inhibits its interaction with PDK1. Our studies further provide a mechanistic model to explain different steps in the docking interaction and regulation. Interestingly, we found that the mechanism that negatively regulates the docking interaction of PRK2 to the upstream kinase PDK1 is directly linked to the activation mechanism of PRK2 itself. Finally, our results indicate that the mechanisms underlying the regulation of the interaction between PRK2 and PDK1 are specific for PRK2 and do not apply for other AGC kinases.
Project description: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.
Project description:Bacterial speck disease in tomato is caused by Pseudomonas syringae pv. tomato. Resistance to this disease is conferred by the host Pto kinase, which recognizes P. s. pv. tomato strains that express the effector AvrPto. We report here that an AvrPto-dependent Pto-interacting protein 3 (Adi3) is a member of the AGC family of protein kinases. In mammals, AGC kinases are regulated by 3-phosphoinositide-dependent protein kinase-1 (Pdk1). We characterized tomato Pdk1 and showed that Pdk1 and Pto phosphorylate Adi3. Gene silencing of Adi3 in tomato causes MAPKKKalpha-dependent formation of necrotic lesions. Use of a chemical inhibitor of Pdk1, OSU-03012, also implicates Pdk1 and Adi3 in plant cell death regulation. Adi3 thus appears to function analogously to the mammalian AGC kinase protein kinase B/Akt by negatively regulating cell death via Pdk1 phosphorylation. We speculate that the negative regulatory function of Adi3 might be subverted by interaction with Pto/AvrPto, leading to host cell death that is associated with pathogen attack.
Project description:Phosphoinositide-dependent protein kinase 1 (PDK1) plays a central role in cellular signaling by phosphorylating members of the AGC family of kinases. This family includes protein kinase C (PKC), protein kinase B (PKB), p70/p90 ribosomal S6 kinases (RSK and S6K), and the catalytic subunit of cAMP-dependent protein kinase (PKA). Although PDK1 phosphorylates and activates PKC, PKB, and RSK in vivo, PDK1 regulation of PKA remains controversial. We isolated ksg1, the fission yeast ortholog of mammalian PDK1, as a suppressor of growth defects caused by loss of the stress-activated MAP kinase, Spc1. Here, we demonstrate that Ksg1 is required for activation of PKA. Cells containing the ksg1.12 thermolabile allele exhibit pleiotropic phenotypes, including the failure to arrest in G(1) and an inability to conjugate. The ksg1.12 allele strongly suppresses defects associated with unregulated PKA. Pka1, the catalytic subunit of cAMP-dependent protein kinase, is phosphorylated in vivo at Thr-356, which is located in the activation loop of the kinase and corresponds to Thr-197 in mammalian PKA. Phosphorylation of Thr-356 is required for in vivo activation of Pka1 and is dependent upon Ksg1. These data provide experimental evidence that PKA is a physiological substrate for PDK1.
Project description:N-alpha-tosyl-L-phenylalanyl chloromethyl ketone (TPCK) has anti-tumorigenic properties, but its direct cellular targets are unknown. Previously, we showed TPCK inhibited the PDKl-dependent AGC kinases RSK, Akt and S6K1 without inhibiting PKA, ERK1/2, PI3K, and PDK1 itself. Here we show TPCK-inhibition of the RSK-related kinases MSK1 and 2, which can be activated independently of PDK1. Mass spectrometry analysis of RSK1, Aktl, S6K1 and MSK1 immunopurified from TPCK-treated cells identified TPCK adducts on cysteines located in conserved activation loop Phenylalanine-Cysteine (Phe-Cys) motifs. Mutational analysis of the Phe-Cys residues conferred partial TPCK resistance. These studies elucidate a primary mechanism by which TPCK inhibits several AGC kinases, inviting consideration of TPCK-like compounds in chemotherapy given their potential for broad control of cellular growth, proliferation and survival.
Project description:The growth factor/insulin-stimulated AGC kinases share an activation mechanism based on three phosphorylation sites. Of these, only the role of the activation loop phosphate in the kinase domain and the hydrophobic motif (HM) phosphate in a C-terminal tail region are well characterized. We investigated the role of the third, so-called turn motif phosphate, also located in the tail, in the AGC kinases PKB, S6K, RSK, MSK, PRK and PKC. We report cooperative action of the HM phosphate and the turn motif phosphate, because it binds a phosphoSer/Thr-binding site above the glycine-rich loop within the kinase domain, promoting zipper-like association of the tail with the kinase domain, serving to stabilize the HM in its kinase-activating binding site. We present a molecular model for allosteric activation of AGC kinases by the turn motif phosphate via HM-mediated stabilization of the alphaC helix. In S6K and MSK, the turn motif phosphate thereby also protects the HM from dephosphorylation. Our results suggest that the mechanism described is a key feature in activation of upto 26 human AGC kinases.
Project description:Phosphoinositide-dependent kinase l (PDK1) phosphorylates and activates multiple AGC serine kinases, including protein kinase B (PKB), p70Ribosomal S6 kinase (S6K) and p90Ribosomal S6 kinase (RSK). PDK1 is required for thymocyte differentiation and proliferation, and herein, we explore the molecular basis for these essential functions of PDK1 in T lymphocyte development. A key finding is that PDK1 is required for the expression of key nutrient receptors in T cell progenitors: CD71 the transferrin receptor and CD98 a subunit of L-amino acid transporters. PDK1 is also essential for Notch-mediated trophic and proliferative responses in thymocytes. A PDK1 mutant PDK1 L155E, which supports activation of PKB but no other AGC kinases, can restore CD71 and CD98 expression in pre-T cells and restore thymocyte differentiation. However, PDK1 L155E is insufficient for thymocyte proliferation. The role of PDK1 in thymus development thus extends beyond its ability to regulate PKB. In addition, PDK1 phosphorylation of AGC kinases such as S6K and RSK is also necessary for thymocyte development.
Project description:Organisms rely heavily on protein phosphorylation to transduce intracellular signals. The phosphorylation of a protein often induces conformational changes, which are responsible for triggering downstream cellular events. Protein kinases are themselves frequently regulated by phosphorylation. Recently, we and others proposed the molecular mechanism by which phosphorylation at a hydrophobic motif (HM) regulates the conformation and activity of many members of the AGC group of protein kinases. Here we have developed specific, low molecular weight compounds, which target the HM/PIF-pocket and have the ability to allosterically activate phosphoinositide-dependent protein kinase 1 (PDK1) by modulating the phosphorylation-dependent conformational transition. The mechanism of action of these compounds was characterized by mutagenesis of PDK1, synthesis of compound analogs, interaction-displacement studies and isothermal titration calorimetry experiments. Our results raise the possibility of developing drugs that target the AGC kinases via a novel mode of action and may inspire future rational development of compounds with the ability to modulate phosphorylation-dependent conformational transitions in other proteins.