The intrinsic substrate specificity of the human tyrosine kinome.

Phosphorylation of proteins on tyrosine (Tyr) residues evolved in metazoan organisms as a mechanism of coordinating tissue growth1. Multicellular eukaryotes typically have more than 50 distinct protein Tyr kinases that catalyse the phosphorylation of thousands of Tyr residues throughout the proteome1-3. How a given Tyr kinase can phosphorylate a specific subset of proteins at unique Tyr sites is only partially understood4-7. Here we used combinatorial peptide arrays to profile the substrate sequence specificity of all human Tyr kinases. Globally, the Tyr kinases demonstrate considerable diversity in optimal patterns of residues surrounding the site of phosphorylation, revealing the functional organization of the human Tyr kinome by substrate motif preference. Using this information, Tyr kinases that are most compatible with phosphorylating any Tyr site can be identified. Analysis of mass spectrometry phosphoproteomic datasets using this compendium of kinase specificities accurately identifies specific Tyr kinases that are dysregulated in cells after stimulation with growth factors, treatment with anti-cancer drugs or expression of oncogenic variants. Furthermore, the topology of known Tyr signalling networks naturally emerged from a comparison of the sequence specificities of the Tyr kinases and the SH2 phosphotyrosine (pTyr)-binding domains. Finally we show that the intrinsic substrate specificity of Tyr kinases has remained fundamentally unchanged from worms to humans, suggesting that the fidelity between Tyr kinases and their protein substrate sequences has been maintained across hundreds of millions of years of evolution.

In vivo assessment of increased oxidation of branched-chain amino acids in glioblastoma.

Altered branched-chain amino acids (BCAAs) metabolism is a distinctive feature of various cancers and plays an important role in sustaining tumor proliferation and aggressiveness. Despite the therapeutic and diagnostic potentials, the role of BCAA metabolism in cancer and the activities of associated enzymes remain unclear. Due to its pivotal role in BCAA metabolism and rapid cellular transport, hyperpolarized 13C-labeled α-ketoisocaproate (KIC), the α-keto acid corresponding to leucine, can assess both BCAA aminotransferase (BCAT) and branched-chain α-keto acid dehydrogenase complex (BCKDC) activities via production of [1-13C]leucine or 13CO2 (and thus H13CO3-), respectively. Here, we investigated BCAA metabolism of F98 rat glioma model in vivo using hyperpolarized 13C-KIC. In tumor regions, we observed a decrease in 13C-leucine production from injected hyperpolarized 13C-KIC via BCAT compared to the contralateral normal-appearing brain, and an increase in H13CO3-, a catabolic product of KIC through the mitochondrial BCKDC. A parallel ex vivo 13C NMR isotopomer analysis following steady-state infusion of [U-13C]leucine to glioma-bearing rats verified the increased oxidation of leucine in glioma tissue. Both the in vivo hyperpolarized KIC imaging and the leucine infusion study indicate that KIC catabolism is upregulated through BCAT/BCKDC and further oxidized via the citric acid cycle in F98 glioma.

Development of Dihydroxyphenyl Sulfonylisoindoline Derivatives as Liver-Targeting Pyruvate Dehydrogenase Kinase Inhibitors.

Pyruvate dehydrogenase kinases 1-4 (PDK1-4) negatively control activity of the pyruvate dehydrogenase complex (PDC) and are up-regulated in obesity, diabetes, heart failure, and cancer. We reported earlier two novel pan-PDK inhibitors PS8 [4-((5-hydroxyisoindolin-2-yl)sulfonyl)benzene-1,3-diol] (1) and PS10 [2-((2,4-dihydroxyphenyl)sulfonyl)isoindoline-4,6-diol] (2) that targeted the ATP-binding pocket in PDKs. Here, we developed a new generation of PDK inhibitors by extending the dihydroxyphenyl sulfonylisoindoline scaffold in 1 and 2 to the entrance region of the ATP-binding pocket in PDK2. The lead inhibitor (S)-3-amino-4-(4-((2-((2,4-dihydroxyphenyl)sulfonyl)isoindolin-5-yl)amino)piperidin-1-yl)-4-oxobutanamide (17) shows a ∼8-fold lower IC50 (58 nM) than 2 (456 nM). In the crystal structure, the asparagine moiety in 17 provides additional interactions with Glu-262 from PDK2. Treatment of diet-induced obese mice with 17 resulted in significant liver-specific augmentation of PDC activity, accompanied by improved glucose tolerance and drastically reduced hepatic steatosis. These findings support 17 as a potential glucose-lowering therapeutic targeting liver for obesity and type 2 diabetes.

Pathogenic mechanisms underlying X-linked Charcot-Marie-Tooth neuropathy (CMTX6) in patients with a pyruvate dehydrogenase kinase 3 mutation.

Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral neuropathy. An X-linked form of CMT (CMTX6) is caused by a missense mutation (R158H) in the pyruvate dehydrogenase kinase isoenzyme 3 (PDK3) gene. PDK3 is one of 4 isoenzymes that negatively regulate the activity of the pyruvate dehydrogenase complex (PDC) by reversible phosphorylation of its first catalytic component pyruvate dehydrogenase (designated as E1). Mitochondrial PDC catalyses the oxidative decarboxylation of pyruvate to acetyl CoA and links glycolysis to the energy-producing Krebs cycle. We have previously shown the R158H mutation confers PDK3 enzyme hyperactivity. In this study we demonstrate that the increased PDK3 activity in patient fibroblasts (PDK3(R158H)) leads to the attenuation of PDC through hyper-phosphorylation of E1 at selected serine residues. This hyper-phosphorylation can be reversed by treating the PDK3(R158H) fibroblasts with the PDK inhibitor dichloroacetate (DCA). In the patient cells, down-regulation of PDC leads to increased lactate, decreased ATP and alteration of the mitochondrial network. Our findings highlight the potential to develop specific drug targeting of the mutant PDK3 as a therapeutic approach to treating CMTX6.

Simultaneous steady-state and dynamic 13C NMR can differentiate alternative routes of pyruvate metabolism in living cancer cells.

Metabolic reprogramming facilitates cancer cell growth, so quantitative metabolic flux measurements could produce useful biomarkers. However, current methods to analyze flux in vivo provide either a steady-state overview of relative activities (infusion of (13)C and analysis of extracted metabolites) or a dynamic view of a few reactions (hyperpolarized (13)C spectroscopy). Moreover, although hyperpolarization has successfully quantified pyruvate-lactate exchanges, its ability to assess mitochondrial pyruvate metabolism is unproven in cancer. Here, we combined (13)C hyperpolarization and isotopomer analysis to quantify multiple fates of pyruvate simultaneously. Two cancer cell lines with divergent pyruvate metabolism were incubated with thermally polarized [3-(13)C]pyruvate for several hours, then briefly exposed to hyperpolarized [1-(13)C]pyruvate during acquisition of NMR spectra using selective excitation to maximize detection of H[(13)C]O3(-) and [1-(13)C]lactate. Metabolites were then extracted and subjected to isotopomer analysis to determine relative rates of pathways involving [3-(13)C]pyruvate. Quantitation of hyperpolarized H[(13)C]O3(-) provided a single definitive metabolic rate, which was then used to convert relative rates derived from isotopomer analysis into quantitative fluxes. This revealed that H[(13)C]O3(-) appearance reflects activity of pyruvate dehydrogenase rather than pyruvate carboxylation followed by subsequent decarboxylation reactions. Glucose substantially altered [1-(13)C]pyruvate metabolism, enhancing exchanges with [1-(13)C]lactate and suppressing H[(13)C]O3(-) formation. Furthermore, inhibiting Akt, an oncogenic kinase that stimulates glycolysis, reversed these effects, indicating that metabolism of pyruvate by both LDH and pyruvate dehydrogenase is subject to the acute effects of oncogenic signaling on glycolysis. The data suggest that combining (13)C isotopomer analyses and dynamic hyperpolarized (13)C spectroscopy may enable quantitative flux measurements in living tumors.

Structure-guided development of specific pyruvate dehydrogenase kinase inhibitors targeting the ATP-binding pocket.

Pyruvate dehydrogenase kinase isoforms (PDKs 1-4) negatively regulate activity of the mitochondrial pyruvate dehydrogenase complex by reversible phosphorylation. PDK isoforms are up-regulated in obesity, diabetes, heart failure, and cancer and are potential therapeutic targets for these important human diseases. Here, we employed a structure-guided design to convert a known Hsp90 inhibitor to a series of highly specific PDK inhibitors, based on structural conservation in the ATP-binding pocket. The key step involved the substitution of a carbonyl group in the parent compound with a sulfonyl in the PDK inhibitors. The final compound of this series, 2-[(2,4-dihydroxyphenyl)sulfonyl]isoindoline-4,6-diol, designated PS10, inhibits all four PDK isoforms with IC50 = 0.8 µM for PDK2. The administration of PS10 (70 mg/kg) to diet-induced obese mice significantly augments pyruvate dehydrogenase complex activity with reduced phosphorylation in different tissues. Prolonged PS10 treatments result in improved glucose tolerance and notably lessened hepatic steatosis in the mouse model. The results support the pharmacological approach of targeting PDK to control both glucose and fat levels in obesity and type 2 diabetes.

Zinc-dependent interaction between JAB1 and pre-S2 mutant large surface antigen of hepatitis B virus and its implications for viral hepatocarcinogenesis.

Chronic hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC) worldwide. The pre-S2 mutant large HBV surface protein (Δ2 LHBS), which contains an in-frame deletion of approximately 17 amino acids in LHBS, is highly associated with risks and prognoses of HBV-induced HCC. It was previously reported that Δ2 LHBS interacts with the Jun activation domain-binding protein 1 (JAB1), a zinc metalloprotease. This promotes the degradation of the cell cycle regulator p27(Kip1) and is believed to be the major mechanism for Δ2 LHBS-induced HCC. In this study, it was found that the interaction between JAB1 and Δ2 LHBS is facilitated by divalent metal Zn(2+) ions. The binding of JAB1 to Δ2 LHBS requires the JAB1/CSN5 MPN metalloenzyme (JAMM) motif and residue H138 that binds to Zn(2+) ions in JAB1. Isothermal titration calorimetry showed that Δ2 LHBS binds directly to Zn(2+) ions in a two-site binding mode. Residues H71 and H116 in Δ2 LHBS, which also contact Zn(2+) ions, are also indispensable for Δ2 LHBS-mediated p27(Kip1) degradation in human HuH7 cells. These results suggest that developing drugs that interrupt interactions between Δ2 LHBS and JAB1 can be used to mitigate Δ2 LHBS-associated risks for HCC.

A new locus for X-linked dominant Charcot-Marie-Tooth disease (CMTX6) is caused by mutations in the pyruvate dehydrogenase kinase isoenzyme 3 (PDK3) gene.

Hereditary motor and sensory disorders of the peripheral nerve form one of the most common groups of human genetic diseases collectively called Charcot-Marie-Tooth (CMT) neuropathy. Using linkage analysis in a three generation kindred, we have mapped a new locus for X-linked dominant CMT to chromosome Xp22.11. A microsatellite scan of the X chromosome established significant linkage to several markers including DXS993 (Zmax = 3.16; θ = 0.05). Extended haplotype analysis refined the linkage region to a 1.43-Mb interval flanked by markers DXS7110 and DXS8027. Whole exome sequencing identified a missense mutation c.G473A (p.R158H) in the pyruvate dehydrogenase kinase isoenzyme 3 (PDK3) gene. The change localized within the 1.43-Mb linkage interval, segregated with the affected phenotype and was excluded in ethnically matched control chromosomes. PDK3 is one of the four isoenzymes regulating the pyruvate dehydrogenase complex (PDC), by reversible phosphorylation, and is a nuclear-coded protein located in the mitochondrial matrix. PDC catalyzes the oxidative decarboxylation of pyruvate to acetyl CoA and is a key enzyme linking glycolysis to the energy-producing Krebs cycle and lipogenic pathways. We found that the R158H mutation confers enzyme hyperactivity and binds with stronger affinity than the wild-type to the inner-lipoyl (L2) domain of the E2p chain of PDC. Our findings suggest a reduced pyruvate flux due to R158H mutant PDK3-mediated hyper-phosphorylation of the PDC as the underlying pathogenic cause of peripheral neuropathy. The results highlight an important causative link between peripheral nerve degeneration and an essential bioenergetic or biosynthetic pathway required for the maintenance of peripheral nerves.

Pivotal role of the C-terminal DW-motif in mediating inhibition of pyruvate dehydrogenase kinase 2 by dichloroacetate.

The mitochondrial pyruvate dehydrogenase complex (PDC) is down-regulated by phosphorylation catalyzed by pyruvate dehydrogenase kinase (PDK) isoforms 1-4. Overexpression of PDK isoforms and therefore reduced PDC activity prevails in cancer and diabetes. In the present study, we investigated the role of the invariant C-terminal DW-motif in inhibition of human PDK2 by dichloroacetate (DCA). Substitutions were made in the DW-motif (Asp-382 and Trp-383) and its interacting residues (Tyr-145 and Arg-149) in the other subunit of PDK2 homodimer. Single and double mutants show 20-60% residual activities that are not stimulated by the PDC core. The R149A and Y145F/R149A mutants show drastic increases in apparent IC(50) values for DCA, whereas binding affinities for DCA are comparable with wild-type PDK2. Both R149A and Y145F variants exhibit increased similar affinities for ADP and ATP, mimicking the effects of DCA. The R149A and the DW-motif mutations (D382A/W383A) forestall binding of the lipoyl domain of PDC to these mutants, analogous to wild-type PDK2 in the presence of DCA and ADP. In contrast, the binding of a dihydrolipoamide mimetic AZD7545 is largely unaffected in these PDK2 variants. Our results illuminate the pivotal role of the DW-motif in mediating communications between the DCA-, the nucleotide-, and the lipoyl domain-binding sites. This signaling network locks PDK2 in the inactive closed conformation, which is in equilibrium with the active open conformation without DCA and ADP. These results implicate the DW-motif anchoring site as a drug target for the inhibition of aberrant PDK activity in cancer and diabetes.

Pyruvate dehydrogenase kinase-4 structures reveal a metastable open conformation fostering robust core-free basal activity.

Human pyruvate dehydrogenase complex (PDC) is down-regulated by pyruvate dehydrogenase kinase (PDK) isoforms 1-4. PDK4 is overexpressed in skeletal muscle in type 2 diabetes, resulting in impaired glucose utilization. Here we show that human PDK4 has robust core-free basal activity, which is considerably higher than activity levels of other PDK isoforms stimulated by the PDC core. PDK4 binds the L3 lipoyl domain, but its activity is not significantly stimulated by any individual lipoyl domains or the core of PDC. The 2.0-A crystal structures of the PDK4 dimer with bound ADP reveal an open conformation with a wider active-site cleft, compared with that in the closed conformation epitomized by the PDK2-ADP structure. The open conformation in PDK4 shows partially ordered C-terminal cross-tails, in which the conserved DW (Asp(394)-Trp(395)) motif from one subunit anchors to the N-terminal domain of the other subunit. The open conformation fosters a reduced binding affinity for ADP, facilitating the efficient removal of product inhibition by this nucleotide. Alteration or deletion of the DW-motif disrupts the C-terminal cross-tail anchor, resulting in the closed conformation and the nearly complete inactivation of PDK4. Fluorescence quenching and enzyme activity data suggest that compounds AZD7545 and dichloroacetate lock PDK4 in the open and the closed conformational states, respectively. We propose that PDK4 with bound ADP exists in equilibrium between the open and the closed conformations. The favored metastable open conformation is responsible for the robust basal activity of PDK4 in the absence of the PDC core.

An expanded conformation of single-ring GroEL-GroES complex encapsulates an 86 kDa substrate.

Electron cryomicroscopy reveals an unprecedented conformation of the single-ring mutant of GroEL (SR398) bound to GroES in the presence of Mg-ATP. This conformation exhibits a considerable expansion of the folding cavity, with approximately 80% more volume than the X-ray structure of the equivalent cis cavity in the GroEL-GroES-(ADP)(7) complex. This expanded conformation can encapsulate an 86 kDa heterodimeric (alphabeta) assembly intermediate of mitochondrial branched-chain alpha-ketoacid dehydrogenase, the largest substrate ever observed to be cis encapsulated. The SR398-GroES-Mg-ATP complex is found to exist as a mixture of standard and expanded conformations, regardless of the absence or presence of the substrate. However, the presence of even a small substrate causes a pronounced bias toward the expanded conformation. Encapsulation of the large assembly intermediate is supported by a series of electron cryomicroscopy studies as well as the protection of both alpha and beta subunits of the substrate from tryptic digestion.

Structural determinants for cross-talk between pyruvate dehydrogenase kinase 3 and lipoyl domain 2 of the human pyruvate dehydrogenase complex.

Pyruvate dehydrogenase kinase isoforms (PDK1-4) are the molecular switch that down-regulates activity of the human pyruvate dehydrogenase complex through reversible phosphorylation. We showed previously that binding of the lipoyl domain 2 (L2) of the pyruvate dehydrogenase complex to PDK3 induces a "cross-tail" conformation in PDK3, resulting in an opening of the active site cleft and the stimulation of kinase activity. In the present study, we report that alanine substitutions of Leu-140, Glu-170, and Glu-179 in L2 markedly reduce binding affinities of these L2 mutants for PDK3. Unlike wildtype L2, binding of these L2 mutants to PDK3 does not preferentially reduce the affinity of PDK3 for ADP over ATP. The inefficient removal of product inhibition associated with ADP accounts for the decreased stimulation of PDK3 activity by these L2 variants. Serial truncations of the PDK3 C-terminal tail region either impede or abolish the binding of wild-type L2 to the PDK3 mutants, resulting in the reduction or absence of L2-enhanced kinase activity. Alanine substitutions of residues Leu-27, Phe-32, Phe-35, and Phe-48 in the lipoyl-binding pocket of PDK3 similarly nullify L2 binding and L2-stimulated PDK3 activity. Our results indicate that the above residues in L2 and residues in the C-terminal region and the lipoyl-binding pocket of PDK3 are critical determinants for the cross-talk between L2 and PDK3, which up-regulates PDK3 activity.

Crystal structure of pyruvate dehydrogenase kinase 3 bound to lipoyl domain 2 of human pyruvate dehydrogenase complex.

The human pyruvate dehydrogenase complex (PDC) is regulated by reversible phosphorylation by four isoforms of pyruvate dehydrogenase kinase (PDK). PDKs phosphorylate serine residues in the dehydrogenase (E1p) component of PDC, but their amino-acid sequences are unrelated to eukaryotic Ser/Thr/Tyr protein kinases. PDK3 binds to the inner lipoyl domains (L2) from the 60-meric transacetylase (E2p) core of PDC, with concomitant stimulated kinase activity. Here, we present crystal structures of the PDK3-L2 complex with and without bound ADP or ATP. These structures disclose that the C-terminal tail from one subunit of PDK3 dimer constitutes an integral part of the lipoyl-binding pocket in the N-terminal domain of the opposing subunit. The two swapped C-terminal tails promote conformational changes in active-site clefts of both PDK3 subunits, resulting in largely disordered ATP lids in the ADP-bound form. Our structural and biochemical data suggest that L2 binding stimulates PDK3 activity by disrupting the ATP lid, which otherwise traps ADP, to remove product inhibition exerted by this nucleotide. We hypothesize that this allosteric mechanism accounts, in part, for E2p-augmented PDK3 activity.