The c-Kit proto-oncogene is a receptor protein-tyrosine kinase associated with several highly malignant human cancers. Upon binding its ligand, stem cell factor (SCF), c-Kit forms an active dimer that autophosphorylates itself and activates a signaling cascade that induces cell growth. Disease-causing human mutations that activate SCF-independent constitutive expression of c-Kit are found in acute myelogenous leukemia, human mast cell disease, and gastrointestinal stromal tumors. We report on the phosphorylation state and crystal structure of a c-Kit product complex. The c-Kit structure is in a fully active form, with ordered kinase activation and phosphate-binding loops. These results provide key insights into the molecular basis for c-Kit kinase transactivation to assist in the design of new competitive inhibitors targeting activated mutant forms of c-Kit that are resistant to current chemotherapy regimes.Receptor protein-tyrosine kinases (RPTKs) 1 regulate key signal transduction cascades that control cellular growth and proliferation. The stem cell factor (SCF) receptor c-Kit is a type III transmembrane RPTK comprised of five extracellular immunoglobulin domains, a single transmembrane region, an inhibitory cytoplasmic juxtamembrane domain, and a split cytoplasmic kinase domain separated by a kinase insert segment (1, 2). The type III RPTK family includes c-Kit (3), the colonystimulating factor-1 (formerly FMS) (4), the platelet-derived growth factor ␣ and ␤ receptors (1, 5), and the FMS-related receptor FLT-3 (6). Signaling by RPTKs occurs via ligand binding to the extracellular IG domains, inducing the receptors to form dimers, and thereby activating intrinsic tyrosine kinase activity through the transphosphorylation of specific tyrosine residues in the juxtamembrane and kinase domains (7,8). Ligand binding both activates kinase activity and creates tyrosine-phosphorylated receptors that mediate the specific binding of intracellular signaling proteins. Src homology 2 and protein tyrosine binding domains (9), including the proteintyrosine phosphatase SHP-1, act as negative regulators of c-Kit activity (10). These cytoplasmic signaling proteins initiate serine/threonine phosphorylation cascades that activate transcription factors to determine specific cellular responses (Fig. 1).The human c-Kit gene is the cellular homologue of the v-kit oncogene found in the transforming Hardy-Zuckerman 4 feline sarcoma virus (11) and encodes a 976-amino acid residue RPTK. Loss-of-function c-Kit mutations establish its importance for the normal growth of hematopoietic progenitor cells, mast cells, melanocytes, primordial germ cells, and the interstitial cells of Cajal (12-15). Gain-of-function mutations, resulting in SCF-independent, constitutive activation of c-Kit, are found in several highly malignant cancers. Mutations in the c-Kit juxtamembrane region cluster around the two main autophosphorylation sites that mediate protein tyrosine binding, Tyr-568 and Tyr-570, and are associated with human gastrointestinal stromal t...

A continuous assay for pp60c-src tyrosine kinase (srcTK) was developed. A lag in phosphorylation of the peptide RRLIEDAEYAARG was observed that could be eliminated by preincubation with MgATP. The induction time for this lag was dependent upon MgATP and srcTK concentrations. When autophosphorylation was monitored by 32P incorporation from [gamma-32P]ATP, a lag in the time course was also observed. These results demonstrate that autoactivation is an intermolecular process. The electrospray ionization mass spectrum of the enzyme before and after activation demonstrated an increase in the phosphorylation state of the enzyme after incubation with MgATP. The delta 85-N-terminal mutant protein and a full-length G2A pp60c-src mutant, which removes the myristylation site, used in these studies were partially phosphorylated on Y338 and Y530 as isolated. This is the first report of phosphorylation on Y338, but the significance of this site of phosphorylation is unknown. These phosphorylations were insufficient to active the enzyme for transfer of the gamma-phosphoryl of MgATP to the peptides. The unphosphorylated enzyme initially present was converted to a monophosphorylated species upon treatment with MgATP. Y-419 phosphorylation was evident only after treatment with MgATP. These data are consistent with autophosphorylation on Y-419 as predicted. Intermolecular autophosphorylation is consistent with the ability of srcTK to dimerize, which is analogous to activation of receptor tyrosine kinases such as the EGF receptor kinase in response to growth factors. These results indicate that dimerization leading to activation does not require binding to the membrane or a hydrophobic N-terminus in the case of srcTK.

Alogliptin is a potent, selective inhibitor of the serine protease dipeptidyl peptidase IV (DPP-4). Herein, we describe the structure-based design and optimization of alogliptin and related quinazolinone-based DPP-4 inhibitors. Following an oral dose, these noncovalent inhibitors provide sustained reduction of plasma DPP-4 activity and a lowering of blood glucose in animal models of diabetes. Alogliptin is currently undergoing phase III trials in patients with type 2 diabetes.

018. Figure 4 depicts a recently obtained cocrystal structure (PDB code 2ONC).

An automated parallel analytical/preparative HPLC/MS workstation has been developed to increase the analysis speed for characterizing and purifying combinatorial libraries. The system incorporates two columns operated in parallel for both LC/MS analysis and preparative LC/ MS purifications (a technique we have named "parallel Analyt/PrepLCMS"). Keys to the successful development of this system have been the design of a multiple sprayer IonSpray interface to support flows from multiple columns and the design of a valving configuration which is under complete software control for delivering the crude samples to the two HPLC columns from a single autosampler. In comparison with the conventional LC/MS systems, where compounds are analyzed sequentially (i.e., one at a time), our parallel Analyt/PrepLCMS system provides an important increase in sample throughput, especially critical in the field of combinatorial chemistry, where compound libraries are generated frequently in parallel arrays and require high-throughout analysis techniques. Minor hardware modifications to the HPLC system were made to facilitate parallel chromatography analyses. By incorporating fast chromatography and mass-based fraction collection on our new parallel Analyt/PrepLCMS system, we demonstrate that more than 200 compounds can be characterized per instrument per day, and more than 200 compounds can be purified per instrument per night. The concept, design, and applications of parallel HPLC/MS to combinatorial libraries are presented and discussed. Salmon, S. E.; Hersh, E. M.; Hraby, V. J.; Kazmiersjum, W. M.; Knapp, R. J. Nature 1991, 354, 82-84. (2) Furka, A.; Sobestyen, F.; Asgedom, M.; Dibo, G. Int. J. Pept. Protein Res. 1991, 37, 487-493. (3) An, H. Y.; Cummins, L. L.; Griffey, R. H.; Bharadwai, R.; Haly, B. D.; Fraser, A. S.; Wilsonlingardo, L.; Risen, L. M.; Wyatt, J. R.; Cook, P. D.

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A rapid method for identifying and characterizing sites of phosphorylation of peptides and proteins is described. High-performance capillary liquid chromatography (HPLC) coupled with electrospray ionization mass spectrometry (ESI-MS) is used to distinguish non-phosphorylated and phosphorylated peptides originating from mixtures as complex as enzyme digests. The method relies on the ability to produce a fragment ion characteristic and unique to phosphopeptides (m/z 79, PO3) by stepping the orifice potential of the mass spectrometer as a function of mass. At low m/z values, a high orifice potential is applied to induce extensive fragmentation of the peptide, leading to the formation of the m/z 79 phosphate-derived ion. This method is analogous to that described by Carr et al. for the identification of glycopeptides from enzymatic digestion of glycoproteins (S.A. Carr, M.J. Huddleston, M.F. Bean, Protein Science 2, 183 (1993)). The method was first evaluated and validated for a mixture of non-, mono- and di-phosphorylated synthetic peptides. Both mono- and di-phosphorylated peptides were found to generate fragment ions characteristic of PO3 whereas the non-phosphorylated peptide did not. Application of the method was extended to identifying phosphopeptides generated from an endoprotease Lys-C digestion of beta-casein. Both the expected mono- and tetra-phosphorylated Lys-C peptides were observed and identified rapidly in the LC/SEI-MS analysis. The procedure was used additionally to identify the site(s) of phosphorylation of the cytosolic non-receptor tyrosine kinase, pp60(c-src).

Stathmin is a regulator of microtubule dynamics which undergoes extensive phosphorylation during the cell cycle as well as in response to various extracellular factors. Four serine residues are targets for protein kinases: Ser-25 and Ser-38 for proline-directed kinases such as mitogen-activated protein kinase and cyclin-dependent protein kinase, and Ser-16 and Ser-63 for cAMP-dependent protein kinase. We studied the effect of phosphorylation on the microtubule-destabilizing activity of stathmin and on its interaction with tubulin in vitro. We show that triple phosphorylation on Ser-16, Ser-25, and Ser-38 efficiently inhibits its activity and prevents its binding to tubulin.