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.     

Disruption of 3-phosphoinositide-dependent kinase 1 (PDK1) signaling by the anti-tumorigenic and anti-proliferative agent n-alpha-tosyl-l-phenylalanyl chloromethyl ketone

The anti-tumorigenic and anti-proliferative effects of N-alpha-tosyl-l-phenylalanyl chloromethyl ketone (TPCK) have been known for more than three decades. Yet little is known about the discrete cellular targets of TPCK controlling these effects. Previous work from our laboratory showed TPCK, like the immunosuppressant rapamycin, to be a potent inhibitor of the 70-kilodalton ribosomal S6 kinase 1 (S6K1), which mediates events involved in cell growth and proliferation. We show here that rapamycin and TPCK display distinct inhibitory mechanisms on S6K1 as a rapamycin-resistant form of S6K1 was TPCK-sensitive. Additionally, we show that TPCK inhibited the activation of the related kinase and proto-oncogene Akt. Upstream regulators of S6K1 and Akt include phosphoinositide 3-kinase (PI 3-K) and 3-phosphoinositide-dependent kinase 1 (PDK1). Whereas TPCK had no effect on either mitogen-regulated PI 3-K activity or total cellular PDK1 activity, TPCK prevented phosphorylation of the PDK1 regulatory sites in S6K1 and Akt. Furthermore, whereas both PDK1 and the mitogen-activated protein kinase (MAPK) are required for full activation of the 90-kilodalton ribosomal S6 kinase (RSK), TPCK inhibited RSK activation without inhibiting MAPK activation. Consistent with the capacity of RSK and Akt to mediate a cell survival signal, in part through phosphorylation of the pro-apoptotic protein BAD, TPCK reduced BAD phosphorylation and led to cell death in interleukin-3-dependent 32D cells. Finally, in agreement with results seen in embryonic stem cells lacking PDK1, protein kinase A activation was not inhibited by TPCK showing TPCK specificity for mitogen-regulated PDK1 signaling. TPCK inhibition of PDK1 signaling thus disables central kinase cascades governing diverse cellular processes including proliferation and survival and provides an explanation for its striking biological effects.     

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.     

3-磷酸肌醇依赖性蛋白激酶1结构和功能

PDK1可调节AGC激酶家族中一些重要蛋白激酶。这些激酶包括蛋白激酶B（PKB／Akt）、p70核小体S6激酶（p70 ribosomal S6 kinase,S6K）、血清和糖皮质激素诱导激酶（SGK）和蛋白激酶C（PKC）等,它们在细胞代谢、生长、增殖和存活等生理过程中具有重要作用。因此,了解PDK1生物学特性可能对其调节的AGC激酶持续活化的癌症治疗具有一定推动作用。本文对PDK1的结构、遗传和生化特点进行了综述。     

Regulation of catalytic activity of S6 kinase 2 during cell cycle

Ribosomal S6 kinase 2 (S6K2) is one of the kinases regulated by the mammalian target of rapamycin (mTOR) signaling pathway. Although it has been identified as a kinase homologous to S6K1, evidence suggests that the two kinases have non-overlapping functions, and the biological function of S6K2 still remains unknown. In order to identify the cell cycle stage(s) during which S6K2 plays a role, we assessed changes in the catalytic activity of S6K2 throughout the cell cycle. Our data show that S6K2 is active throughout the cell cycle with higher activity in G2 and M phases. We also show that S6K1 activity peaks sharply during M phase. Our data suggest that S6K1 and S6K2 likely play yet-unknown roles in G2 and M phases.     

A phosphoserine-regulated docking site in the protein kinase RSK2 that recruits and activates PDK1

The 90 kDa ribosomal S6 kinase-2 (RSK2) is a growth factor-stimulated protein kinase with two kinase domains. The C-terminal kinase of RSK2 is activated by ERK-type MAP kinases, leading to autophosphorylation of RSK2 at Ser386 in a hydrophobic motif. The N-terminal kinase is activated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) through phosphorylation of Ser227, and phosphorylates the substrates of RSK. Here, we identify Ser386 in the hydrophobic motif of RSK2 as a phosphorylation-dependent docking site and activator of PDK1. Treatment of cells with growth factor induced recruitment of PDK1 to the Ser386-phosphorylated hydrophobic motif and phosphorylation of RSK2 at Ser227. A RSK2-S386K mutant showed no interaction with PDK1 or phosphorylation at Ser227. Interaction with Ser386-phosphorylated RSK2 induced autophosphorylation of PDK1. Addition of a synthetic phosphoSer386 peptide (RSK2(373-396)) increased PDK1 activity 6-fold in vitro. Finally, mutants of RSK2 and MSK1, a RSK-related kinase, with increased affinity for PDK1, were constitutively active in vivo and phosphorylated histone H3. Our results suggest a novel regulatory mechanism based on phosphoserine-mediated recruitment of PDK1 to RSK2, leading to coordinated phosphorylation and activation of PDK1 and RSK2.     

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.     

Structural insights into the regulation of PDK1 by phosphoinositides and inositol phosphates

3-phosphoinositide-dependent protein kinase-1 (PDK1) phosphorylates and activates many kinases belonging to the AGC subfamily. PDK1 possesses a C-terminal pleckstrin homology (PH) domain that interacts with PtdIns(3,4,5)P3/PtdIns(3,4)P2 and with lower affinity to PtdIns(4,5)P2. We describe the crystal structure of the PDK1 PH domain, in the absence and presence of PtdIns(3,4,5)P3 and Ins(1,3,4,5)P4. The structures reveal a 'budded' PH domain fold, possessing an N-terminal extension forming an integral part of the overall fold, and display an unusually spacious ligand-binding site. Mutagenesis and lipid-binding studies were used to define the contribution of residues involved in phosphoinositide binding. Using a novel quantitative binding assay, we found that Ins(1,3,4,5,6)P5 and InsP6, which are present at micromolar levels in the cytosol, interact with full-length PDK1 with nanomolar affinities. Utilising the isolated PDK1 PH domain, which has reduced affinity for Ins(1,3,4,5,6)P5/InsP6, we perform localisation studies that suggest that these inositol phosphates serve to anchor a portion of cellular PDK1 in the cytosol, where it could activate its substrates such as p70 S6-kinase and p90 ribosomal S6 kinase that do not interact with phosphoinositides.     

Characterization of GSK2334470, a novel and highly specific inhibitor of PDK1

PDK1 (3-phosphoinositide-dependent protein kinase 1) activates a group of protein kinases belonging to the AGC [PKA (protein kinase A)/PKG (protein kinase G)/PKC (protein kinase C)]-kinase family that play important roles in mediating diverse biological processes. Many cancer-driving mutations induce activation of PDK1 targets including Akt, S6K (p70 ribosomal S6 kinase) and SGK (serum- and glucocorticoid-induced protein kinase). In the present paper, we describe the small molecule GSK2334470, which inhibits PDK1 with an IC?? of ~10 nM, but does not suppress the activity of 93 other protein kinases including 13 AGC-kinases most related to PDK1 at 500-fold higher concentrations. Addition of GSK2334470 to HEK (human embryonic kidney)-293, U87 or MEF (mouse embryonic fibroblast) cells ablated T-loop residue phosphorylation and activation of SGK isoforms and S6K1 induced by serum or IGF1 (insulin-like growth factor 1). GSK2334470 also inhibited T-loop phosphorylation and activation of Akt, but was more efficient at inhibiting Akt in response to stimuli such as serum that activated the PI3K (phosphoinositide 3-kinase) pathway weakly. GSK2334470 inhibited activation of an Akt1 mutant lacking the PH domain (pleckstrin homology domain) more potently than full-length Akt1, suggesting that GSK2334470 is more effective at inhibiting PDK1 substrates that are activated in the cytosol rather than at the plasma membrane. Consistent with this, GSK2334470 inhibited Akt activation in knock-in embryonic stem cells expressing a mutant of PDK1 that is unable to interact with phosphoinositides more potently than in wild-type cells. GSK2334470 also suppressed T-loop phosphorylation and activation of RSK2 (p90 ribosomal S6 kinase 2), another PDK1 target activated by the ERK (extracellular-signal-regulated kinase) pathway. However, prolonged treatment of cells with inhibitor was required to observe inhibition of RSK2, indicating that PDK1 substrates possess distinct T-loop dephosphorylation kinetics. Our data define how PDK1 inhibitors affect AGC signalling pathways and suggest that GSK2334470 will be a useful tool for delineating the roles of PDK1 in biological processes.     

In vivo activation of protein kinase A in Schizosaccharomyces pombe requires threonine phosphorylation at its activation loop and is dependent on PDK1

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.     

3-磷酸肌醇依赖性蛋白激酶-1激活AGC家族激酶的机制研究进展

3磷酸肌醇依赖性蛋白激酶1(3phosphoinositidedependentproteinkinase1,PDK1PDPK1)是蛋白激酶B(proteinkinaseB,PKBCAKT)的上游激酶,通过与3,4,5三磷酸磷脂酰肌醇[PtdIns(3,4,5)P3]作用激活相邻的PKB分子.同时,PDK1被称为AGC激酶的掌管者(master),能够激活包含PKB在内的一系列的AGC激酶家族成员.PDK1磷酸化这些激酶的保守区域Tloop区,使它们充分激活,从而调节细胞代谢,生长,扩散,生存,抗凋亡等诸多生理过程.本文就PDK1调节AGC激酶的活性,与功能上命名的PDK2的关系,PDK1分子自身的调节,PH结构域对自身活性及AGC激酶活性的影响,PDK1定位以及作为一个新药物靶标等方面做了综述.     

Identification of S6K2 as a centrosome-located kinase

Ribosomal S6 kinase 2 (S6K2) acts downstream of the mammalian target of rapamycin (mTOR). Here, we show that some S6K2 localize at the centrosome throughout the cell cycle. S6K2 is found in the pericentriolar area of the centrosome. S6K2 centrosomal localization is unaffected by serum withdrawal or treatment with rapamycin, wortmannin, U0126, or phorbol-12-myristate-13-acetate (PMA). Unlike S6K2, S6 kinase 1 (S6K1) does not localize at the centrosome, suggesting the two kinases may also have nonoverlapping functions. Our data suggest that centrosomal S6K2 may have a role in the phosphoinositide-3-kinase (PI3K)/Akt/mTOR signaling pathway that has also been detected in the centrosome.     

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.     

Phosphorylation of human microtubule-associated protein tau by protein kinases of the AGC subfamily

Intraneuronal inclusions made of hyperphosphorylated microtubule-associated protein tau are a defining neuropathological characteristic of Alzheimer's disease, and of several other neurodegenerative disorders. Many phosphorylation sites in tau are S/TP sites that flank the microtubule-binding repeats. Others are KXGS motifs in the repeats. One site upstream of the repeats lies in a consensus sequence for AGC kinases. This site (S214) is believed to play an important role in the events leading from normal, soluble to filamentous, insoluble tau. Here, we show that all AGC kinases tested phosphorylated S214. RSK1 and p70 S6 kinase also phosphorylated the neighbouring T212, a TP site that conforms weakly to the AGC kinase consensus sequence. MSK1 phosphorylated S214, as well as S262, a KXGS site in the first repeat, and S305 in the second repeat.     

Phosphoinositide-dependent protein kinase-1 (PDK1)-independent activation of the protein kinase C substrate, protein kinase D

Phosphoinoisitide dependent kinase l (PDK1) is proposed to phosphorylate a key threonine residue within the catalytic domain of the protein kinase C (PKC) superfamily that controls the stability and catalytic competence of these kinases. Hence, in PDK1-null embryonic stem cells intracellular levels of PKCalpha, PKCbeta1, PKCgamma, and PKCepsilon are strikingly reduced. Although PDK1-null cells have reduced endogenous PKC levels they are not completely devoid of PKCs and the integrity of downstream PKC effector pathways in the absence of PDK1 has not been determined. In the present report, the PDK1 requirement for controlling the phosphorylation and activity of a well characterised substrate for PKCs, the serine kinase protein kinase D, has been examined. The data show that in embryonic stem cells and thymocytes loss of PDK1 does not prevent PKC-mediated phosphorylation and activation of protein kinase D. These results reveal that loss of PDK1 does not functionally inactivate all PKC-mediated signal transduction.     

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).     

90-kDa ribosomal S6 kinase is phosphorylated and activated by 3-phosphoinositide-dependent protein kinase-1.

90-kDa ribosomal S6 kinase-2 (RSK2) belongs to a family of growth factor-activated serine/threonine kinases composed of two kinase domains connected by a regulatory linker region. The N-terminal kinase of RSK2 is involved in substrate phosphorylation. Its activation requires phosphorylation of the linker region at Ser(369), catalyzed by extracellular signal-regulated kinase (ERK), and at Ser(386), catalyzed by the C-terminal kinase, after its activation by ERK. In addition, the N-terminal kinase must be phosphorylated at Ser(227) in the activation loop by an as yet unidentified kinase. Here, we show that the isolated N-terminal kinase of RSK2 (amino acids 1-360) is phosphorylated at Ser(227) by PDK1, a constitutively active kinase, leading to 100-fold stimulation of kinase activity. In COS7 cells, ectopic PDK1 induced the phosphorylation of full-length RSK2 at Ser(227) and Ser(386), without involvement of ERK, leading to partial activation of RSK2. Similarly, two other members of the RSK family, RSK1 and RSK3, were partially activated by PDK1 in COS7 cells. Finally, our data indicate that full activation of RSK2 by growth factor requires the cooperation of ERK and PDK1 through phosphorylation of Ser(227), Ser(369), and Ser(386). Our study extend recent findings which implicate PDK1 in the activation of protein kinases B and C and p70(S6K), suggesting that PDK1 controls several major growth factor-activated signal transduction pathways.     

环境变化和时序衰老过程中酵母蛋白激酶Sch9磷酸化的调控

出芽酵母(Saccharomyces cerevisiae)蛋白激酶Sch9与哺乳动物蛋白激酶S6K1同源.S6K1是哺乳动物雷帕霉素靶蛋白(mTOR)和磷脂酰肌醇3激酶(PI3K)的底物,且与很多人类疾病相关,包括肥胖症、糖尿病和癌症.Sch9和S6K1都对不同营养条件和环境胁迫条件下的细胞生长调控很重要.Sch9激活环内的磷酸化位点570位苏氨酸残基也被称为PDK1位点,而737位苏氨酸位点也被称为PDK2位点,这两个位点的磷酸化对Sch9的活性非常重要.蛋白激酶Pkh1/2磷酸化Sch9的PDK1位点,而雷帕霉素靶蛋白复合体1(TORC1)磷酸化PDK2位点.为了深入了解Sch9在细胞中的功能,阐明不同环境条件下及时序衰老过程中Sch9的PDK1和PDK2位点磷酸化的调控就显得尤为重要.利用特异性识别570位苏氨酸残基磷酸化的Sch9蛋白和特异性识别737位苏氨酸残基磷酸化的Sch9蛋白的两种抗体,对不同环境条件下和时序衰老过程中Sch9的两个位点的磷酸化调控进行了研究.研究结果揭示了Sch9的两个磷酸化位点在营养感受、胁迫应答、热量限制和时序衰老过程中的调控方式.揭示Sch9的PDK1位点磷酸化的调控与热量限制延长出芽酵母时序寿命密切相关.