PDK2: the missing piece in the receptor tyrosine kinase signaling pathway puzzle

Activation of members of the protein kinase AGC (cAMP dependent, cGMP dependent, and protein kinase C) family is regulated primarily by phosphorylation at two sites: a conserved threonine residue in the activation loop and a serine/threonine residue in a hydrophobic motif (HM) near the COOH terminus. Although phosphorylation of these kinases in the activation loop has been found to be mediated by phosphoinositide-dependent protein kinase-1 (PDK1), the kinase(s) that catalyzes AGC kinase phosphorylation in the HM remains uncharacterized. So far, at least 10 kinases have been suggested to function as an HM kinase or the so-called "PDK2," including mitogen-activated protein (MAP) kinase-activated protein kinase-2 (MK2), integrin-linked kinase (ILK), p38 MAP kinase, protein kinase Calpha (PKCalpha), PKCbeta, the NIMA-related kinase-6 (NEK6), the mammalian target of rapamycin (mTOR), the double-stranded DNA-dependent protein kinase (DNK-PK), and the ataxia telangiectasia mutated (ATM) gene product. However, whether any or all of these kinases act as a physiological HM kinase remains to be established. Nonetheless, available data suggest that multiple systems may be used in cells to regulate the activation of the AGC family kinases. It is possible that, unlike activation loop phosphorylation, phosphorylation of the HM site in the different AGC family kinases is mediated by distinct kinases. In addition, phosphorylation of the AGC family kinase at the HM site could be cell type, signaling pathway, and substrate specific. Identification and characterization of the bonafide HM kinase(s) will be essential to verify these hypotheses.     

Regulation of the Interaction between Protein Kinase C-related Protein Kinase 2 (PRK2) and Its Upstream Kinase, 3-Phosphoinositide-dependent Protein Kinase 1 (PDK1)

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 ³²P_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.The regulation of protein function by phosphorylation and dephosphorylation is a key mechanism of intracellular signaling pathways in eukaryotic organisms. Protein phosphorylation is catalyzed by protein kinases, which are themselves often regulated by phosphorylation (1). The specificity of protein kinases is essential for their cellular functions. In some groups of protein kinases, the specificity is achieved by means of “docking interactions.” Protein kinase docking interactions involve a recognition site on the kinase or a flanking domain that is different from the active site. The most notable example, MAP kinases, uses a docking interaction to specifically recognize substrates, upstream kinases, and phosphatases. Despite the large amount of data on protein kinase docking interactions, e.g. in the MAP kinase field, there is very little information on how these essential interactions are regulated (2–4).3-Phosphoinositide-dependent protein kinase 1 (PDK1)³ belongs to the AGC family of protein kinases and is the activation loop kinase for several other AGC kinases (5). A key feature of the AGC kinase family members except PDK1 is the presence of a C-terminal extension (CT) to the catalytic core that contains a conserved hydrophobic motif (HM) harboring a phosphorylation site. In many AGC kinases, the HM mediates a docking interaction with PDK1. For example, p90 ribosomal S6 kinase (RSK), p70 S6 kinase (S6K) and serum- and glucocorticoid-induced protein kinase (SGK) interact with PDK1 upon phosphorylation of the HM site (6–9). The phosphorylated HM binds to a HM-binding pocket in the catalytic core of PDK1 that was originally termed the PIF-binding pocket (6, 10).Besides its role in the docking of substrates to PDK1, the HM/PIF-binding pocket was also identified as a ubiquitous and key regulatory site in likely all AGC kinases (7, 11). Thus, in AGC kinases studied up to now, the HM/PIF-binding pocket serves as an intramolecular docking site for the phosphorylated HM. In summary, the HM has a dual function in AGC kinase activation, (i) mediating the intermolecular interaction with PDK1 and (ii) acting as an intramolecular allosteric activator that stabilizes the active conformation of the kinase domain via binding to the HM/PIF-binding pocket.The CT of AGC kinases additionally contains a second regulatory phosphorylation site traditionally termed the “turn motif” (TM), and more recently the zipper (Z) site. The Z/TM phosphate interacts with a binding site within the kinase domain, acting like a zipper which serves to support the intramolecular binding of the phosphorylated HM to the HM/PIF-binding pocket (12). Hence, AGC kinases are synergistically activated by phosphorylation at the activation loop, the HM, and the Z/TM sites.Protein kinase C-related protein kinases (PRKs) (13) (also named PAK for protease-activated kinase (14–16) and PKN for protein kinase N (17)) represent a subfamily of AGC kinases. So far, three PRK isoforms were identified, PRK1, PRK2, and PKN3, which are effectors of the small GTP-binding protein Rho. PRKs, as well as the Rho-associated kinases (ROCKs), are considered to be the protein kinases that mediate the phosphorylation events downstream of Rho activation and both can be inhibited by the highly specific protein kinase inhibitor Y27632 (18). The most notable role described for PRK2 is the control of entry into mitosis and exit from cytokinesis (19). In addition, PRK2 phosphorylates the hepatitis C virus (HCV) RNA polymerase (20). In support of a function in HCV RNA replication, PRK2 inhibitors like Y27632 suppress HCV replication (21).The N-terminal region of PRK2 possesses three Rho effector (HR1) domains (13), a pseudosubstrate region that is thought to have an autoinhibitory function (22) and a C2-like domain, which is a potential binding site for lipid activators. The C-terminal region of PRK2 harbors the HM that mediates the docking interaction with the HM/PIF-binding pocket in its upstream kinase PDK1 (10, 23). Interestingly, PRKs and also atypical protein kinase Cs (PKCs, PKCζ, and PKCι/λ), contain an acidic residue instead of a phosphorylatable amino acid at the site equivalent to the HM phosphorylation site in other AGC kinases. Therefore, the molecular events that regulate the interaction of PRK2 and PKCζ with PDK1 must be different from the mechanism characterized for S6K, SGK, and RSK.In the present work we extended and refined the model of docking interaction between PRK2 and PDK1 and characterized C-terminal regions of PRK2 that participate in the regulation of this interaction. The work sheds light on the common as well as specific mechanisms that operate in the regulation of PDK1 docking interaction with its different substrates.     

Notch-induced T cell development requires phosphoinositide-dependent kinase 1

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.     

Fine tuning PDK1 activity by phosphorylation at Ser163

3-Phosphoinositide-dependent protein kinase-1 (PDK1) mediates phosphorylation and activation of members of the AGC protein kinase family and plays an essential role in insulin signaling and action. However, whether and how PDK1 activity is regulated in cells remains largely uncharacterized. In the present study, we show that PDK1 undergoes insulin-stimulated and phosphatidylinositol 3-kinase-dependent phosphorylation at Ser244 in the activation loop and at a novel site: Ser163 in the hinge region between the two lobes of the kinase domain. Sequence alignment studies revealed that the residue corresponding to Ser163 of PDK1 in all other AGC kinases is glutamate, suggesting that a negative charge at this site may be important for PDK1 function. Replacing Ser163 with a negatively charged residue, glutamate, led to a 2-fold increase in PDK1 activity. Molecular modeling studies suggested that phosphorylated Ser163 may form additional hydrogen bonds with Tyr149 and Gln223. In support of this, mutation of Tyr149 to Ala is sufficient to reduce PDK1 activity. Taken together, our results suggest that PDK1 phosphorylation of Ser163 may provide a mechanism to fine-tune PDK1 activity and function in cells.     

Phosphoinositide-dependent protein kinase 1, a sensor of protein conformation

Phosphoinositide-dependent protein kinase 1 (PDK1) is a protein kinase that phosphorylates and activates several other protein kinases from the AGC group (which includes PKA, PKG and PKC), to which PDK1 also belongs. Recent data suggests that PDK1 specificity is achieved by regulation of its interaction with substrates and supports a rather simple model explaining how PDK1 interacts with different substrates. The data further suggests that PDK1 interacts with its substrates when they are in a particular conformation (inactive). PDK1 has the ability to recognize, interact with and phosphorylate specific substrate conformations and thus sets PDK1 at the centre of a protein conformation sensor mechanism. The PDK1-substrate interaction model describes, at a molecular level, the mechanism used by PDK1 to sense the conformation of its substrates.     

Role of T-loop phosphorylation in PDK1 activation, stability, and substrate binding

3-Phosphoinositide-dependent protein kinase-1 (PDK1) phosphorylates the T-loop of several AGC (cAMP-dependent, cGMP-dependent, protein kinase C) family protein kinases, resulting in their activation. Previous structural studies have revealed that the alpha C-helix, located in the small lobe of the kinase domain of PDK1, is a key regulatory element, as it links a substrate interacting site termed the hydrophobic motif (HM) pocket with the phosphorylated Ser-241 in the T-loop. In this study we have demonstrated by mutational analysis that interactions between the phosphorylated Ser-241 and the alpha C-helix are not required for PDK1 activity or substrate binding through the HM-pocket but are necessary for PDK1 to be activated or stabilized by a peptide that binds to this site. The structure of an inactive T-loop mutant of PDK1, in which Ser-241 is changed to Ala, was also determined. This structure, together with surface plasmon resonance binding studies, demonstrates that the PDK1(S241A)-inactive mutant possesses an intact HM-pocket as well as an ordered alpha C-helix. These findings reveal that the integrity of the alpha C-helix and HM-pocket in PDK1 is not regulated by T-loop phosphorylation.     

AGC protein kinases: From structural mechanism of regulation to allosteric drug development for the treatment of human diseases

The group of AGC protein kinases includes more than 60 protein kinases in the human genome, classified into 14 families: PDK1, AKT/PKB, SGK, PKA, PKG, PKC, PKN/PRK, RSK, NDR, MAST, YANK, DMPK, GRK and SGK494. This group is also widely represented in other eukaryotes, including causative organisms of human infectious diseases. AGC kinases are involved in diverse cellular functions and are potential targets for the treatment of human diseases such as cancer, diabetes, obesity, neurological disorders, inflammation and viral infections. Small molecule inhibitors of AGC kinases may also have potential as novel therapeutic approaches against infectious organisms. Fundamental in the regulation of many AGC kinases is a regulatory site termed the “PIF-pocket” that serves as a docking site for substrates of PDK1. This site is also essential to the mechanism of activation of AGC kinases by phosphorylation and is involved in the allosteric regulation of N-terminal domains of several AGC kinases, such as PKN/PRKs and atypical PKCs. In addition, the C-terminal tail and its interaction with the PIF-pocket are involved in the dimerization of the DMPK family of kinases and may explain the molecular mechanism of allosteric activation of GRKs by GPCR substrates. In this review, we briefly introduce the AGC kinases and their known roles in physiology and disease and the discovery of the PIF-pocket as a regulatory site in AGC kinases. Finally, we summarize the current status and future therapeutic potential of small molecules directed to the PIF-pocket; these molecules can allosterically activate or inhibit the kinase as well as act as substrate-selective inhibitors. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).     

Expression, purification, and characterization of a structurally disordered and functional C-terminal autoinhibitory domain (AID) of the 70 kDa 40S ribosomal protein S6 kinase-1 (S6K1)

S6K1 is a member of the AGC subfamily of serine-threonine protein kinases, whereby catalytic activation requires dual phosphorylation of critical residues in the conserved T-loop (T229) and hydrophobic motif (HM; T389) peptide regions of its catalytic kinase domain (residues 1-398). In addition to its kinase domain, S6K1 contains a C-terminal autoinhibitory domain (AID; residues 399-502), which prevents T-loop and HM phosphorylation; and autoinhibition is relieved on multi-site Ser-Thr phosphorylation of the AID (S411, S418, T421, and S424). Interestingly, 66 of the 104 C-terminal AID amino acid residues were computer predicted to exist in structurally disordered peptide regions, begetting interest as to how such dynamics could be coupled to autoregulation. To begin addressing this issue, we developed and optimized protocols for efficient AID expression and purification. Consistent with computer predictions, aberrant mobilities in both SDS-PAGE and size-exclusion chromatography, as well as low chemical shift dispersion in (1)H-(15)N HSQC NMR spectra, indicated purified recombinant AID to be largely unfolded. Yet, trans-addition of purified AID effectively inhibited PDK1-catalyzed T-loop phosphorylation of a catalytic kinase domain construct of S6K1. Using an identical purification protocol, similar protein yields of a tetraphospho-mimic mutant AID(D(2)ED) construct were obtained; and this construct displayed only weak inhibition of PDK1-catalyzed T229 phosphorylation. Purification of the structurally 'disordered' and functional C-terminal AID and AID(D(2)ED) constructs will facilitate studies aimed to understand the role of conformational plasticity and protein phosphorylation in modulating autoregulatory domain-domain interactions.     

A chimeric mechanism for polyvalent trans‐phosphorylation of PKA by PDK1

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)_1‐2Y/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.     

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 PKCzeta, 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 PKCzeta and PRK2 by phosphorylating the T-loop residue. In the present study we demonstrate that the hydrophobic motifs of PKCzeta and PKCiota, 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 PKCzeta, 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 PKCzeta. These findings indicate that the hydrophobic motif of PRK2 and PKCzeta acts as a "docking site" enabling the recruitment of PDK1 to these substrates. This is essential for their phosphorylation by PDK1 in cells.     

Multiple implications of 3-phosphoinositide-dependent protein kinase 1 in human cancer

3-phosphoinositide-dependent protein kinase-1 (PDK1) is a central mediator of cellular signaling between phosphoinositide-3 kinase and various intracellular serine/threonine kinases, including protein kinase B, p70 ribosomal S6 kinase, serum and glucocorticoid-inducible kinase, and protein kinase C. PDK1 activates members of the AGC family of protein kinases by phosphorylating serine/threonine residues in the activation loop. Here, we review the regulatory mechanisms of PDK1 and its roles in cancer. PDK1 is activated by autophosphorylation in the activation loop and other serine residues, as well as by phosphorylation of Tyr-9 and Tyr-373/376. Src appears to recognize PDK1 following tyrosine phosphorylation. The role of heat shock protein 90 in regulating PDK1 stability and PDK1-Src complex formation are also discussed. Furthermore, we summarize the subcellular distribution of PDK1. Finally, an important role for PDK1 in cancer chemotherapy is proposed. In conclusion, a better understanding of its molecular regulatory mechanisms in various signaling pathways will help to explain how PDK1 acts as an oncogenic kinase in various cancers, and will contribute to the development of novel cancer chemotherapies.     

A phosphoserine/threonine-binding pocket in AGC kinases and PDK1 mediates activation by hydrophobic motif phosphorylation

The growth factor-activated AGC protein kinases RSK, S6K, PKB, MSK and SGK are activated by serine/threonine phosphorylation in the activation loop and in the hydrophobic motif, C-terminal to the kinase domain. In some of these kinases, phosphorylation of the hydrophobic motif creates a specific docking site that recruits and activates PDK1, which then phosphorylates the activation loop. Here, we discover a pocket in the kinase domain of PDK1 that recognizes the phosphoserine/phosphothreonine in the hydrophobic motif by identifying two oppositely positioned arginine and lysine residues that bind the phosphate. Moreover, we demonstrate that RSK2, S6K1, PKBalpha, MSK1 and SGK1 contain a similar phosphate-binding pocket, which they use for intramolecular interaction with their own phosphorylated hydrophobic motif. Molecular modelling and experimental data provide evidence for a common activation mechanism in which the phosphorylated hydrophobic motif and activation loop act on the alphaC-helix of the kinase structure to induce synergistic stimulation of catalytic activity. Sequence conservation suggests that this mechanism is a key feature in activation of >40 human AGC kinases.     

Regulation of protein kinases in insulin,growth factor and Wnt signalling

Protein kinase cascades provide the regulatory mechanisms for many of the essential processes in eukaryotic cells. Recent structural and biochemical work has revealed the basis of phosphorylation regulation of three consecutive protein kinases - phosphoinositide-dependent kinase 1 (PDK1), protein kinase B (PKB)/Akt and glycogen synthase kinase 3beta (GSK3beta) - which transduce signals generated by insulin and/or growth factors binding to cell surface receptors. PDK1 and PKB are both AGC family kinases. Whereas PKB is positively regulated via its phosphorylated C-terminal hydrophobic motif, the activity and specificity of PDK1 are determined by equivalent hydrophobic motifs of substrate AGC kinases. In a contrasting mechanism, GSK3beta is negatively regulated by competitive autoinhibition by its phosphorylated N terminus. GSK3beta also functions in the developmental Wnt signalling pathway, but without cross-talk with the PDK1-PKB/Akt pathway. Structural studies of GSK3beta complexes are contributing to our understanding of the phosphorylation-independent mechanism that insulates the Wnt and insulin/growth factor pathways.     

Mechanism for activation of the growth factor-activated AGC kinases by turn motif phosphorylation

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.     

Analysis of 3-phosphoinositide-dependent kinase-1 signaling and function in ES cells

3-phosphoinositide-dependent kinase-1 (PDK1) phosphorylates and activates several kinases in the cAMP-dependent, cGMP-dependent and protein kinase C (AGC) family. Many putative PDK1 substrates have been identified, but have not been analyzed following transient and specific inhibition of PDK1 activity. Here, we demonstrate that a previously characterized PDK1 inhibitor, BX-795, shows biological effects that are not consistent with PDK1 inhibition. Therefore, we describe the creation and characterization of a PDK1 mutant, L159G, which can bind inhibitor analogues containing bulky groups that hinder access to the ATP binding pocket of wild type (WT) kinases. When expressed in PDK1(-/-) ES cells, PDK1 L159G restored phosphorylation of PDK1 targets known to be hypophosphorylated in these cells. Screening of multiple inhibitor analogues showed that 1-NM-PP1 and 3,4-DMB-PP1 optimally inhibited the phosphorylation of PDK1 targets in PDK1(-/-) ES cells expressing PDK1 L159G but not WT PDK1. These compounds confirmed previously assumed PDK1 substrates, but revealed distinct dephosphorylation kinetics. While PDK1 inhibition had little effect on cell growth, it sensitized cells to apoptotic stimuli. Furthermore, PDK1 loss abolished growth of allograft tumors. Taken together we describe a model system that allows for acute and reversible inhibition of PDK1 in cells, to probe biochemical and biological consequences.     

Characterization of testis-specific serine-threonine kinase 3 and its activation by phosphoinositide-dependent kinase-1-dependent signalling

The family of testis-specific serine-threonine kinases (TSSKs) consists of four members whose expression is confined almost exclusively to testis. Very little is known about their physiological role and mechanisms of action. We cloned human and mouse TSSK3 and analysed the biochemical properties, substrate specificity and in vitro activation. In vitro TSSK3 exhibited the ability to autophosphorylate and to phosphorylate test substrates such as histones, myelin basic protein and casein. Interestingly, TSSK3 showed maximal in vitro kinase activity at 30 degrees C, in keeping with it being testis specific. Sequence comparison indicated the existence of a so-called 'T-loop' within the TSSK3 catalytic domain, a structure present in the AGC family of protein kinases. To test if this T-loop is engaged in TSSK3 regulation, we mutated the critical threonine residue within the T-loop to alanine (T168A) which resulted in inactivation of TSSK3 kinase. Furthermore, Thr168 is phosphorylated in vitro by the T-loop kinase phosphoinositide-dependent protein kinase-1 (PDK1). PDK1-induced phosphorylation increased in vitro TSSK3 kinase activity, suggesting that TSSK3 can be regulated in the same way as AGC kinase family members. Analysis of peptide sequences identifies the peptide sequence RRSSSY containing Ser5 that is a target for TSSK3 phosphorylation, as an efficient and specific substrate for TSSK3.     

Characterization of a PDK1 homologue from the moss Physcomitrella patens

The serine/threonine protein kinase 3-phosphoinositide-dependent protein kinase 1 (PDK1) is a highly conserved eukaryotic kinase that is a central regulator of many AGC kinase subfamily members. Through its regulation of AGC kinases, PDK1 controls many basic cellular processes, from translation to cell survival. While many of these PDK1-regulated processes are conserved across kingdoms, it is not well understood how PDK1 may have evolved within kingdoms. In order to better understand PDK1 evolution within plants, we have isolated and characterized the PDK1 gene from the moss Physcomitrella patens (PpPDK1), a nonvascular representative of early land plants. PpPDK1 is similar to other plant PDK1s in that it can functionally complement a yeast PDK1 knockout line. However, unlike PDK1 from other plants, the P. patens PDK1 protein does not bind phospholipids due to a lack of the lipid-binding pleckstrin homology domain, which is used for lipid-mediated regulation of PDK1 activity. Sequence analysis of several PDK1 proteins suggests that lipid regulation of PDK1 may not commonly occur in algae and nonvascular land plants. PpPDK1 can phosphorylate AGC kinase substrates from tomato (Solanum lycopersicum) and P. patens at the predicted PDK1 phosphorylation site, indicating that the PpPDK1 substrate phosphorylation site is conserved with higher plants. We have also identified residues within the PpPDK1 kinase domain that affect kinase activity and show that a mutant with highly reduced kinase activity can still confer cell viability in both yeast and P. patens. These studies lay the foundation for further analysis of the evolution of PDK1 within plants.     

Perspectives in PDK1 evolution: Insights from photosynthetic and non-photosynthetic organisms

Protein kinases belonging to the AGC group modulate many diverse cellular processes in all eukaryotes. One important way to regulate AGC kinases is through phosphorylation by the upstream kinase PDK1. PDK1 localization and activity usually depend on interactions with phospholipids, which are mediated by a conserved lipid-binding pleckstrin homology (PH) domain. We recently analyzed putative PDK1 sequences from 17 photosynthetic organisms, finding that PDK1s from vascular and nonvascular species seem to be distinguished by the presence or absence of a PH domain, respectively. The only other reported PDK1 lacking a PH domain is from yeast (Saccharomyces cerevisiae). These observations raise questions about how plant PDK1s and their lipid-binding capabilities have evolved in relation to other eukaryotes, and what this means for PDK1 function. Here we use 100 PDK1 sequences from diverse organisms to discuss possible evolutionary aspects of plant PDK1 structure and lipid binding.     

The role of 3-phosphoinositide-dependent protein kinase 1 in activating AGC kinases defined in embryonic stem cells

BACKGROUND: Protein kinase B (PKB), and the p70 and p90 ribosomal S6 kinases (p70 S6 kinase and p90 Rsk, respectively), are activated by phosphorylation of two residues, one in the 'T-loop' of the kinase domain and, the other, in the hydrophobic motif carboxy terminal to the kinase domain. The 3-phosphoinositide-dependent protein kinase 1 (PDK1) activates many AGC kinases in vitro by phosphorylating the T-loop residue, but whether PDK1 also phosphorylates the hydrophobic motif and whether all other AGC kinases are substrates for PDK1 is unknown. RESULTS: Mouse embryonic stem (ES) cells in which both copies of the PDK1 gene were disrupted were viable. In PDK1(-/-) ES cells, PKB, p70 S6 kinase and p90 Rsk were not activated by stimuli that induced strong activation in PDK1(+/+) cells. Other AGC kinases - namely, protein kinase A (PKA), the mitogen- and stress-activated protein kinase 1 (MSK1) and the AMP-activated protein kinase (AMPK) - had normal activity or were activated normally in PDK1(-/-) cells. The insulin-like growth factor 1 (IGF1) induced PKB phosphorylation at its hydrophobic motif, but not at its T-loop residue, in PDK1(-/-) cells. IGF1 did not induce phosphorylation of p70 S6 kinase at its hydrophobic motif in PDK1(-/-) cells. CONCLUSIONS: PDK1 mediates activation of PKB, p70 S6 kinase and p90 Rsk in vivo, but is not rate-limiting for activation of PKA, MSK1 and AMPK. Another kinase phosphorylates PKB at its hydrophobic motif in PDK1(-/-) cells. PDK1 phosphorylates the hydrophobic motif of p70 S6 kinase either directly or by activation of another kinase.     

High resolution crystal structure of the human PDK1 catalytic domain defines the regulatory phosphopeptide docking site

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.