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。     

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.     

Structural basis for the regulation of protein kinase A by activation loop phosphorylation

The catalytic subunit of cAMP-dependent protein kinase (PKA) is a member of the AGC group of protein kinases. Whereas PKA has served as a structural model for the protein kinase superfamily, all previous structures of the catalytic subunit contain a phosphorylated activation loop. To understand the structural effects of activation loop phosphorylation at Thr-197 we used a PKA mutant that does not autophosphorylate at Thr-197. The enzyme crystallized in the apo-state, and the structure was solved to 3.0 ?. The N-lobe is rotated by 18° relative to the wild-type apoenzyme, which illustrates that the enzyme likely exists in a wide range of conformations in solution due to the uncoupling of the N- and C-lobes. Several regions of the protein including the activation loop are disordered in the structure, and there are alternate main chain conformations for the magnesium positioning loop and catalytic loop causing a complete loss of hydrogen bonding between these two active site structural elements. These alterations are reflected in a 20-fold decrease in the apparent phosphoryl transfer rate as measured by pre-steady-state kinetic methods.     

From determination of amino acid residue conformation to reconstruction of the spatial structure of proteins (from the data of nuclear Overhauser enhancement spectroscopy

A new approach to the calculation of the spatial protein structure based on the joint utilization of the theoretical conformational analysis method and nuclear Overhauser enhancement (NOE) spectroscopy data is proposed and verified. The quality in determining various molecule structural parameters is estimated in terms of the expected NOE spectral parameters derived from the X-ray analysis data of the avian pancreatic polypeptide. The proposed approach is shown to correctly determine such structural parameters of protein molecules as local amino acid residue conformations, reciprocal spatial orientation of the C alpha atoms neighbouring along amino acid sequence and reapproached segments of the polypeptide chain. Spatially remote molecule fragments are mainly responsible for the error in determining structural parameters.     

Phosphoprotein analysis using antibodies broadly reactive against phosphorylated motifs

The substrates of most protein kinases remain unknown because of the difficulty tracing signaling pathways and identifying sites of protein phosphorylation. Here we describe a method useful in detecting subclasses of protein kinase substrates. Although the method is broadly applicable to any protein kinase for which a substrate consensus motif has been identified, we illustrate here the use of antibodies broadly reactive against phosphorylated Ser/Thr-motifs typical of AGC kinase substrates. Phosphopeptide libraries with fixed residues corresponding to consensus motifs RXRXXT*/S* (Akt motif) and S*XR (protein kinase C motif) were used as antigens to generate antibodies that recognize many different phosphoproteins containing the fixed motif. Because most AGC kinase members are phosphorylated and activated by phosphoinositide-dependent protein kinase-1 (PDK1), we used PDK1-/- ES cells to profile potential AGC kinase substrates downstream of PDK1. To identify phosphoproteins detected using the Akt substrate antibody, we characterized the antibody binding specificity to generate a specificity matrix useful in predicting antibody reactivity. Using this approach we predicted and then identified a 30-kDa phosphoprotein detected by both Akt and protein kinase C substrate antibodies as S6 ribosomal protein. Phosphospecific motif antibodies offer a new approach to protein kinase substrate identification that combines immunoreactivity data with protein data base searches based upon antibody specificity.     

Structural organization of the fibrinogen molecule

A brief review of the chemical structure of the fibrinogen molecule, its proteolytic fragmentation, physicochemical and functional properties of proteolytic fragments is presented. The previous notions on the structure of the fibrinogen molecule are considered as well as new data obtained by the methods of scanning microcalorimetry, electron microscopy and by other methods which permit suggesting some models for structural organization of the fibrinogen molecule.     

PDK1, the master regulator of AGC kinase signal transduction

The interaction of insulin and growth factors with their receptors on the outside surface of a cell, leads to the activation of phosphatidylinositol 3-kinase (PI 3-kinase) and generation of the phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) second messenger at the inner surface of the cell membrane. One of the most studied signalling events controlled by PtdIns(3,4,5)P3, comprises the activation of a group of AGC family protein kinases, including isoforms of protein kinase B (PKB)/Akt, p70 ribosomal S6 kinase (S6K), serum- and glucocorticoid-induced protein kinase (SGK) and protein kinase C (PKC), which play crucial roles in regulating physiological processes relevant to metabolism, growth, proliferation and survival. Here, we review recent biochemical, genetic and structural studies on the 3-phosphoinositide-dependent protein kinase-1 (PDK1), which phosphorylates and activates the AGC kinase members regulated by PI 3-kinase. We also discuss whether inhibitors of PDK1 might have chemotherapeutic potential in the treatment of cancers in which the PDK1-regulated AGC kinases are constitutively activated.     

Analogous regulatory sites within the alphaC-beta4 loop regions of ZAP-70 tyrosine kinase and AGC kinases

The precise positioning of the flexible C-helix in the catalytic core is a critical step in the activation of most protein kinases. Consequently, the alphaC-beta4 loop, which anchors the C-helix to the catalytic core, is highly conserved and mediates key structural interactions that serve as a hinge for C-helix movement. While these hinge interactions are conserved across diverse eukaryotic protein kinase structures, some families such as AGC kinases diverge from the canonical hinge interactions. This divergence was recently proposed to facilitate an alternative mode of regulation wherein a conserved C-terminal tail interacts with the alphaC-beta4 loop to position the C-helix. Here we show how interactions between the alphaC-beta4 loop and the N-terminal SH2 domain of ZAP-70 tyrosine kinase are mechanistically and functionally analogous to interactions between the alphaC-beta4 loop and the C-terminal tail of AGC kinases. Such cis regulation of protein kinase activity may be a feature of other eukaryotic protein kinase families as well.     

Mammalian TOR signaling to the AGC kinases

The mechanistic (or mammalian) target of rapamycin (mTOR), an evolutionarily conserved protein kinase, orchestrates cellular responses to growth, metabolic and stress signals. mTOR processes various extracellular and intracellular inputs as part of two mTOR protein complexes, mTORC1 or mTORC2. The mTORCs have numerous cellular targets but members of a family of protein kinases, the protein kinase (PK)A/PKG/PKC (AGC) family are the best characterized direct mTOR substrates. The AGC kinases control multiple cellular functions and deregulation of many members of this family underlies numerous pathological conditions. mTOR phosphorylates conserved motifs in these kinases to allosterically augment their activity, influence substrate specificity, and promote protein maturation and stability. Activation of AGC kinases in turn triggers the phosphorylation of diverse, often overlapping, targets that ultimately control cellular response to a wide spectrum of stimuli. This review will highlight recent findings on how mTOR regulates AGC kinases and how mTOR activity is feedback regulated by these kinases. We will discuss how this regulation can modulate downstream targets in the mTOR pathway that could account for the varied cellular functions of mTOR.     

Mammalian TOR signaling to the AGC kinases

The mechanistic (or mammalian) target of rapamycin (mTOR), an evolutionarily conserved protein kinase, orchestrates cellular responses to growth, metabolic and stress signals. mTOR processes various extracellular and intracellular inputs as part of two mTOR protein complexes, mTORC1 or mTORC2. The mTORCs have numerous cellular targets but members of a family of protein kinases, the protein kinase (PK)A/PKG/PKC (AGC) family are the best characterized direct mTOR substrates. The AGC kinases control multiple cellular functions and deregulation of many members of this family underlies numerous pathological conditions. mTOR phosphorylates conserved motifs in these kinases to allosterically augment their activity, influence substrate specificity, and promote protein maturation and stability. Activation of AGC kinases in turn triggers the phosphorylation of diverse, often overlapping, targets that ultimately control cellular response to a wide spectrum of stimuli. This review will highlight recent findings on how mTOR regulates AGC kinases and how mTOR activity is feedback regulated by these kinases. We will discuss how this regulation can modulate downstream targets in the mTOR pathway that could account for the varied cellular functions of mTOR.     

TOR regulation of AGC kinases in yeast and mammals

The TOR (target of rapamycin), an atypical protein kinase, is evolutionarily conserved from yeast to man. Pharmacological studies using rapamycin to inhibit TOR and yeast genetic studies have provided key insights on the function of TOR in growth regulation. One of the first bona fide cellular targets of TOR was the mammalian protein kinase p70 S6K (p70 S6 kinase), a member of a family of kinases called AGC (protein kinase A/protein kinase G/protein kinase C-family) kinases, which include PKA (cAMP-dependent protein kinase A), PKG (cGMP-dependent kinase) and PKC (protein kinase C). AGC kinases are also highly conserved and play a myriad of roles in cellular growth, proliferation and survival. The AGC kinases are regulated by a common scheme that involves phosphorylation of the kinase activation loop by PDK1 (phosphoinositide-dependent kinase 1), and phosphorylation at one or more sites at the C-terminal tail. The identification of two distinct TOR protein complexes, TORC1 (TOR complex 1) and TORC2, with different sensitivities to rapamycin, revealed that TOR, as part of either complex, can mediate phosphorylation at the C-terminal tail for optimal activation of a number of AGC kinases. Together, these studies elucidated that a fundamental function of TOR conserved throughout evolution may be to balance growth versus survival signals by regulating AGC kinases in response to nutrients and environmental conditions. This present review highlights this emerging function of TOR that is conserved from budding and fission yeast to mammals.     

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.     

The protein kinase C inhibitor bisindolyl maleimide 2 binds with reversed orientations to different conformations of protein kinase A

As the key mediators of eukaryotic signal transduction, the protein kinases often cause disease, and in particular cancer, when disregulated. Appropriately selective protein kinase inhibitors are sought after as research tools and as therapeutic drugs; several have already proven valuable in clinical use. The AGC subfamily protein kinase C (PKC) was identified early as a cause of cancer, leading to the discovery of a variety of PKC inhibitors. Despite its importance and early discovery, no crystal structure for PKC has yet been reported. Therefore, we have co-crystallized PKC inhibitor bisindolyl maleimide 2 (BIM2) with PKA variants to study its binding interactions. BIM2 co-crystallized as an asymmetric pair of kinase-inhibitor complexes. In this asymmetric unit, the two kinase domains have different lobe configurations, and two different inhibitor conformers bind in different orientations. One kinase molecule (A) is partially open with respect to the catalytic conformation, the other (B) represents the most open conformation of PKA reported so far. In monomer A, the BIM2 inhibitor binds tightly via an induced fit in the ATP pocket. The indole moieties are rotated out of the plane with respect to the chemically related but planar inhibitor staurosporine. In molecule B a different conformer of BIM2 binds in a reversed orientation relative to the equivalent maleimide atoms in molecule A. Also, a critical active site salt bridge is disrupted, usually indicating the induction of an inactive conformation. Molecular modeling of the clinical phase III PKC inhibitor LY333531 into the electron density of BIM2 reveals the probable binding mechanism and explains selectivity properties of the inhibitor.     

The crystal structure of human placenta growth factor-1 (PlGF-1), an angiogenic protein, at 2.0 A resolution

The angiogenic molecule placenta growth factor (PlGF) is a member of the cysteine-knot family of growth factors. In this study, a mature isoform of the human PlGF protein, PlGF-1, was crystallized as a homodimer in the crystallographic asymmetric unit, and its crystal structure was elucidated at 2.0 A resolution. The overall structure of PlGF-1 is similar to that of vascular endothelial growth factor (VEGF) with which it shares 42% amino acid sequence identity. Based on structural and biochemical data, we have mapped several important residues on the PlGF-1 molecule that are involved in recognition of the fms-like tyrosine kinase receptor (Flt-1, also known as VEGFR-1). We propose a model for the association of PlGF-1 and Flt-1 domain 2 with precise shape complementarity, consider the relevance of this assembly for PlGF-1 signal transduction, and provide a structural basis for altered specificity of this molecule.     

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.     

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).     

Atomic Structure of GRK5 Reveals Distinct Structural Features Novel for G Protein-coupled Receptor Kinases

G protein-coupled receptor kinases (GRKs) are members of the protein kinase A, G, and C families (AGC) and play a central role in mediating G protein-coupled receptor phosphorylation and desensitization. One member of the family, GRK5, has been implicated in several human pathologies, including heart failure, hypertension, cancer, diabetes, and Alzheimer disease. To gain mechanistic insight into GRK5 function, we determined a crystal structure of full-length human GRK5 at 1.8 Å resolution. GRK5 in complex with the ATP analog 5′-adenylyl β,γ-imidodiphosphate or the nucleoside sangivamycin crystallized as a monomer. The C-terminal tail (C-tail) of AGC kinase domains is a highly conserved feature that is divided into three segments as follows: the C-lobe tether, the active-site tether (AST), and the N-lobe tether (NLT). This domain is fully resolved in GRK5 and reveals novel interactions with the nucleotide and N-lobe. Similar to other AGC kinases, the GRK5 AST is an integral part of the nucleotide-binding pocket, a feature not observed in other GRKs. The AST also mediates contact between the kinase N- and C-lobes facilitating closure of the kinase domain. The GRK5 NLT is largely displaced from its previously observed position in other GRKs. Moreover, although the autophosphorylation sites in the NLT are >20 Å away from the catalytic cleft, they are capable of rapid cis-autophosphorylation suggesting high mobility of this region. In summary, we provide a snapshot of GRK5 in a partially closed state, where structural elements of the kinase domain C-tail are aligned to form novel interactions to the nucleotide and N-lobe not previously observed in other GRKs.     

Potent bicyclic inhibitors of malarial cGMP-dependent protein kinase: approaches to combining improvements in cell potency,selectivity and structural novelty

Focussed studies on imidazopyridine inhibitors of Plasmodium falciparum cyclic GMP-dependent protein kinase (PfPKG) have significantly advanced the series towards desirable in vitro property space. LLE-based approaches towards combining improvements in cell potency, key physicochemical parameters and structural novelty are described, and a structure-based design hypothesis relating to substituent regiochemistry has directed efforts towards key examples with well-balanced potency, ADME and kinase selectivity profiles.     

Lining the pockets of kinases and phosphatases

The regulation of the activity of kinases and phosphatases is an essential aspect of intracellular signal transduction. Recently determined structures of AGC protein kinases, including isoforms of PKB, PKC, GRK and ROCK, indicate that occupancy of a hydrophobic pocket in the kinase N-lobe by a segment of the protein immediately C terminal to the kinase domain provides a mechanism for regulating kinase activity. In addition, crystal structures of Aurora-A and Aurora-B, which are closely related to AGC family kinases, in complex with their activators, TPX2 and INCENP, respectively, show how allosteric kinase activation is achieved by the binding of the activator protein to an equivalent hydrophobic pocket. Hence, regulation of kinase activity by analogous interactions is a shared regulatory mechanism of these kinases. Two crystal structures have explained the molecular basis of PKA anchoring through its regulatory subunits by members of the AKAP family of scaffold proteins. AKAPs can also interact directly with protein kinase and phosphatase catalytic domains. The crystal structure of the PP1 catalytic subunit in complex with the targeting subunit MYPT1 indicates that there is also scope for intimate phosphatase regulation by scaffold proteins.     

Crystal structure of low humidity tetragonal lysozyme at 2.1-A resolution. Variability in hydration shell and its structural consequences

Tetragonal crystals of hen egg white lysozyme undergo a reversible transformation, accompanied by loss of water, when the relative humidity of the environment is reduced to about 90%. The structure of the low humidity form has been analyzed, using x-ray data collected at 88% relative humidity, in order to explore the variability in protein hydration caused by a change in the amount of water surrounding the protein molecule and the consequent conformational perturbations in the molecule. The structure has been refined by the restrained least-squares method to an R value of 0.162 for 6269 observed reflections in the 10-2.1-A resolution shell. The refined structure provides interesting examples for the variability in helical parameters, the role of interactions involving side chains and water in the stabilization of secondary structural features, and favorable specific hydration sites. The protein molecule as a whole moves slightly in the low humidity form from its position in the native crystals. The hydration shell tends to move along with the protein. Significant changes, however, occur in the hydration shell. These changes cause structural perturbations in the enzyme molecule, which are most pronounced in regions involved in substrate binding.