Huaibin Chen

Tyr phosphorylation of PDP1 toggles recruitment between ACAT1 and SIRT3 to regulate the pyruvate dehydrogenase complex.

Mitochondrial pyruvate dehydrogenase complex (PDC) is crucial for glucose homeostasis in mammalian cells. The current understanding of PDC regulation involves inhibitory serine phosphorylation of pyruvate dehydrogenase (PDH) by PDH kinase (PDK), whereas dephosphorylation of PDH by PDH phosphatase (PDP) activates PDC. Here, we report that lysine acetylation of PDHA1 and PDP1 is common in epidermal growth factor (EGF)-stimulated cells and diverse human cancer cells. K321 acetylation inhibits PDHA1 by recruiting PDK1, and K202 acetylation inhibits PDP1 by dissociating its substrate PDHA1, both of which are important in promoting glycolysis in cancer cells and consequent tumor growth. Moreover, we identified mitochondrial ACAT1 and SIRT3 as the upstream acetyltransferase and deacetylase, respectively, of PDHA1 and PDP1, while knockdown of ACAT1 attenuates tumor growth. Furthermore, Y381 phosphorylation of PDP1 dissociates SIRT3 and recruits ACAT1 to PDC. Together, hierarchical, distinct posttranslational modifications act in concert to control molecular composition of PDC and contribute to the Warburg effect.

Loss-of-Function Fibroblast Growth Factor Receptor-2 Mutations in Melanoma

We report that 10% of melanoma tumors and cell lines harbor mutations in the fibroblast growth factor receptor 2 (FGFR2) gene. These novel mutations include three truncating mutations and 20 missense mutations occurring at evolutionary conserved residues in FGFR2 as well as among all four FGFRs. The mutation spectrum is characteristic of those induced by UV radiation. Mapping of these mutations onto the known crystal structures of FGFR2 followed by in vitro and in vivo studies show that these mutations result in receptor loss of function through several distinct mechanisms, including loss of ligand binding affinity, impaired receptor dimerization, destabilization of the extracellular domains, and reduced kinase activity. To our knowledge, this is the first demonstration of loss-of-function mutations in a class IV receptor tyrosine kinase in cancer. Taken into account with our recent discovery of activating FGFR2 mutations in endometrial cancer, we suggest that FGFR2 may join the list of genes that play context-dependent opposing roles in cancer. (Mol Cancer Res 2009;7(1):41–54)

Genetic Overlap in Kallmann Syndrome, Combined Pituitary Hormone Deficiency, and Septo-Optic Dysplasia

CONTEXT Kallmann syndrome (KS), combined pituitary hormone deficiency (CPHD), and septo-optic dysplasia (SOD) all result from development defects of the anterior midline in the human forebrain. OBJECTIVE The objective of the study was to investigate whether KS, CPHD, and SOD have shared genetic origins. DESIGN AND PARTICIPANTS A total of 103 patients with either CPHD (n = 35) or SOD (n = 68) were investigated for mutations in genes implicated in the etiology of KS (FGFR1, FGF8, PROKR2, PROK2, and KAL1). Consequences of identified FGFR1, FGF8, and PROKR2 mutations were investigated in vitro. RESULTS Three patients with SOD had heterozygous mutations in FGFR1; these were either shown to alter receptor signaling (p.S450F, p.P483S) or predicted to affect splicing (c.336C>T, p.T112T). One patient had a synonymous change in FGF8 (c.216G>A, p.T72T) that was shown to affect splicing and ligand signaling activity. Four patients with CPHD/SOD were found to harbor heterozygous rare loss-of-function variants in PROKR2 (p.R85G, p.R85H, p.R268C). CONCLUSIONS Mutations in FGFR1/FGF8/PROKR2 contributed to 7.8% of our patients with CPHD/SOD. These data suggest a significant genetic overlap between conditions affecting the development of anterior midline in the human forebrain.

A crystallographic snapshot of tyrosine trans-phosphorylation in action

Tyrosine trans-phosphorylation is a key event in receptor tyrosine kinase signaling, yet, the structural basis for this process has eluded definition. Here, we present the crystal structure of the FGF receptor 2 kinases caught in the act of trans-phosphorylation of Y769, the major C-terminal phosphorylation site. The structure reveals that enzyme- and substrate-acting kinases engage each other through elaborate and specific interactions not only in the immediate vicinity of Y769 and the enzyme active site, but also in regions that are as much of 18 Å away from D626, the catalytic base in the enzyme active site. These interactions lead to an unprecedented level of specificity and precision during the trans-phosphorylation on Y769. Time-resolved mass spectrometry analysis supports the observed mechanism of trans-phosphorylation. Our data provide a molecular framework for understanding the mechanism of action of Kallmann syndrome mutations and the order of trans-phosphorylation reactions in FGFRs. We propose that the salient mechanistic features of Y769 trans-phosphorylation are applicable to trans-phosphorylation of the equivalent major phosphorylation sites in many other RTKs.

Solid-phase N-terminus PEGylation of recombinant human fibroblast growth factor 2 on heparin-sepharose column.

Recombinant fibroblast growth factor-2 (FGF-2) has been extensively studied and used in several clinical applications including wound healing, bone regeneration, and neuroprotection. Poly(ethylene glycol) (PEG) modification of recombinant human FGF-2 (rhFGF-2) in solution phase has been studied to increase the in vivo biostabilities and therapeutic potency. However, the solution-phase strategy is not site-controlled and the products are often not homogeneous due to the generation of multi-PEGylated proteins. In order to increase mono-PEGylated rhFGF-2 level, a novel solid-phase strategy for rhFGF-2 PEGylation is developed. RhFGF-2 proteins were loaded onto a heparin-sepharose column and the PEGylaton reaction was carried out at the N-terminus by PEG20 kDa butyraldehyde through reductive alkylation. The PEGylated rhFGF-2 was purified to near homogeneity by SP sepharose anion-exchange chromatography and the purity was more than 95% with a yield of mono-PEGylated rhFGF-2 of 58.3%, as confirmed by N-terminal sequencing and MALDI-TOF mass spectrometry. In vitro biophysical and biochemical measurements demonstrated that PEGylated rhFGF-2 has an unchanged secondary structure, receptor binding activity, cell proliferation, and MAP kinase stimulating activity, and an improved bio- and thermal stability. Animal assay showed that PEGylated rhFGF-2 has an increased half-life and reduced immunogenicity. Compared to conventional solution-phase PEGylation, the solid-phase PEGylation is advantageous in reaction time, production of mono-PEGylated protein, and improvement of biochemical and biological activity.

Structural Mimicry of A-Loop Tyrosine Phosphorylation by a Pathogenic FGF Receptor 3 Mutation

The K650E gain-of-function mutation in the tyrosine kinase domain of FGF receptor 3 (FGFR3) causes Thanatophoric Dysplasia type II, a neonatal lethal congenital dwarfism syndrome, and when acquired somatically, it contributes to carcinogenesis. In this report, we determine the crystal structure of the FGFR3 kinase domain harboring this pathogenic mutation and show that the mutation introduces a network of intramolecular hydrogen bonds to stabilize the active-state conformation. In the crystal, the mutant FGFR3 kinases are caught in the act of trans-phosphorylation on a kinase insert autophosphorylation site, emphasizing the fact that the K650E mutation circumvents the requirement for A-loop tyrosine phosphorylation in kinase activation. Analysis of this trans-phosphorylation complex sheds light onto the determinants of tyrosine trans-phosphorylation specificity. We propose that the targeted inhibition of this pathogenic FGFR3 kinase may be achievable by small molecule kinase inhibitors that selectively bind the active-state conformation of FGFR3 kinase.

Two FGF Receptor Kinase Molecules Act in Concert to Recruit and Transphosphorylate Phospholipase Cγ

The molecular basis by which receptor tyrosine kinases (RTKs) recruit and phosphorylate Src Homology 2 (SH2) domain-containing substrates has remained elusive. We used X-ray crystallography, NMR spectroscopy, and cell-based assays to demonstrate that recruitment and phosphorylation of Phospholipase Cγ (PLCγ), a prototypical SH2 containing substrate, by FGF receptors (FGFR) entails formation of an allosteric 2:1 FGFR-PLCγ complex. We show that the engagement of pTyr-binding pocket of the cSH2 domain of PLCγ by the phosphorylated tail of an FGFR kinase induces a conformational change at the region past the cSH2 core domain encompassing Tyr-771 and Tyr-783 to facilitate the binding/phosphorylation of these tyrosines by another FGFR kinase in trans. Our data overturn the current paradigm that recruitment and phosphorylation of substrates are carried out by the same RTK monomer in cis and disclose an obligatory role for receptor dimerization in substrate phosphorylation in addition to its canonical role in kinase activation.

Elucidation of a four-site allosteric network in fibroblast growth factor receptor tyrosine kinases

Receptor tyrosine kinase (RTK) signaling is tightly regulated by protein allostery within the intracellular tyrosine kinase domains. Yet the molecular determinants of allosteric connectivity in tyrosine kinase domain are incompletely understood. By means of structural (X-ray and NMR) and functional characterization of pathogenic gain-of-function mutations affecting the FGF receptor (FGFR) tyrosine kinase domain, we elucidated a long-distance allosteric network composed of four interconnected sites termed the ‘molecular brake’, ‘DFG latch’, ‘A-loop plug’, and ‘αC tether’. The first three sites repress the kinase from adopting an active conformation, whereas the αC tether promotes the active conformation. The skewed design of this four-site allosteric network imposes tight autoinhibition and accounts for the incomplete mimicry of the activated conformation by pathogenic mutations targeting a single site. Based on the structural similarity shared among RTKs, we propose that this allosteric model for FGFR kinases is applicable to other RTKs. DOI: http://dx.doi.org/10.7554/eLife.21137.001

Abstract 4733: Activating FGFR2 kinase domain mutations provide resistance to dovitinib (TKI258)

Members of the fibroblast growth factor receptor (FGFR) family are amplified or mutationally activated in a variety of cancers, including breast, endometrial, ovarian, lung, gastric, and bladder cancers, glioblastoma and rhabdomyosarcoma. Consequently FGFRs are attractive therapeutic targets in cancer, with a number of FGFR inhibitors currently progressing through clinical trials. Dovitinib, a lead FGFR kinase inhibitor exhibits activity against FLT3, FGFRs, VEGFRs, and PDGFR, and has demonstrated considerable preclinical activity in cancer models with FGFR activation. Though targeted tyrosine kinase inhibitors (TKIs) have shown dramatic clinical responses, the long-term efficacy of these agents is frequently limited by development of resistance to the targeted agent, often due to mutation of the target kinase. Here we sought to identify the mutational mechanisms of resistance to Dovitinib using a BaF3 cell line screening strategy. The BaF3 cell line is an IL-3 dependent murine pro-B cell line that is commonly employed to model TKI resistant mutations. These cells do not express any FGF ligands or receptors and introduction and activation of FGFRs has been shown to substitute for IL-3 to promote cell proliferation. BaF3 cells transduced with FGFR2 were treated with TKI258 at 5x, 10x, and 15x the cellular IC50 for Dovitinib in these cells. Following clonal selection of Dovitinib resistant cells, the exons encoding the intracellular domain of FGFR2 were sequenced. Mutations in FGFR2 kinase domain were identified in 26 out of 63 (41.2%) resistant clones screened, with an increase in frequency of mutation with increasing selective pressure. Ten distinct Dovitinib-resistant mutations in FGFR2 were identified and subsequently confirmed to result in Dovitinib-resistance and kinase activation. The binding mode of Dovitinib and the mechanisms of action of the resistance mutations were studied using the crystal structures of unphosphorylated and phosphorylated FGFR2Ks. Mutations at N550 and E566 at the kinase hinge/interlobe region are expected to drive the kinase into the active state by disengaging the molecular brake that keeps the kinase in an autoinhibited state. Five additional mutations are also predicted to stabilize the active conformation of the kinase by strengthening a network of hydrophobic interactions between N- and C-terminal lobes of the kinase, termed the hydrophobic spine, that is a hallmark of the active state of the kinase. Hence our biochemical and structural data show that the drug predominantly binds the inactive state of the FGFR2 kinase. Our data have clinical ramifications as they suggest that cancer patients harboring these FGFR2 mutations may not respond to the anti-FGFR activity of Dovitinib. Taken together our study provides the first report of TKI-resistant mutations in FGFR2 and suggests that the active state of the FGFR2 kinase should be targeted for anti-cancer drug discovery. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4733. doi:10.1158/1538-7445.AM2011-4733

Impaired Fibroblast Growth Factor Receptor 1 Signaling as a Cause of Normosmic Idiopathic Hypogonadotropic Hypogonadism

The Harvard Center for Reproductive Endocrine Sciences and the Reproductive Endocrine Unit of the Department of Medicine (T.R., Y.S., L.P., A.M., E.J.-D., V.A.H., A.D., F.J.H., N.P.), Massachusetts General Hospital, and Harvard Center for Reproductive Endocrine Sciences and Brigham and Women’s Hospital (S.X., U.B.K.), Division of Endocrinology, Diabetes, and Hypertension, Boston, Massachusetts 02114; Department of Pharmacology (H.C., J.M., M.M.), New York University School of Medicine, New York, New York 10016; Institute of Human Genetics (R.Q.), University of Newcastle-upon-Tyne, Newcastle NE2 4HH, United Kingdom; Sainte Justin Hospital (G.V.V.), Montreal, Quebec, Canada H3T 1C5; Centre Hospitalier de l’Universite de Montreal and Procrea Cliniques (H.L.), Montreal, Quebec, Canada H2W1T8; Department of Genetics and Metabolism (S.S.), Children’s National Medical Center, Washington, DC 20010; and Biomedicum Helsinki (T.R.), Institute of Biomedicine/Physiology, University of Helsinki, FIN-00014 Helsinki, Finland