Structural Characterization of Autoinhibited C-Met Kinase Produced by Coexpression in Bacteria with Phosphatase

Structural characterization of autoinhibited c-Met kinase produced by coexpression in bacteria with phosphatase Weiru Wang, Adhirai Marimuthu, James Tsai, Abhinav Kumar, Heike I. Krupka, Chao Zhang, Ben Powell, Yoshihisa Suzuki, Hoa Nguyen, Maryam Tabrizizad, Catherine Luu, and Brian L. West* Plexxikon, Inc., 91 Bolivar Drive, Berkeley, CA 94710 Communicated by Sung-Hou Kim, University of California, Berkeley, CA, January 3, 2006 (received for review December 28, 2005) Protein kinases are a large family of cell signaling mediators found that kinase samples produced in bacteria can be hetero- undergoing intensive research to identify inhibitors or modulators geneously autophosphorylated during expression in bacteria, but useful for medicine. As one strategy, small-molecule compounds that coexpression with different phosphatases works to produce that bind the active site with high affinity can be used to inhibit the kinases in an unphosphorylated form (8). In the current study, enzyme activity. X-ray crystallography is a powerful method to we describe in detail the production of the c-Abl, c-Src, and reveal the structures of the kinase active sites, and thus aid in the c-Met kinases using such a system. design of high-affinity, selective inhibitors. However, a limitation c-Met is the membrane receptor for hepatocyte growth factor still exists in the ability to produce purified kinases in amounts (HGF), and is important for liver development and regeneration sufficient for crystallography. Furthermore, kinases exist in differ- (ref. 9, and references therein). A link between c-Met and cancer ent conformation states as part of their normal regulation, and the was made when it was first cloned as an oncogene, later found ability to prepare crystals of kinases in these various states also to be a truncated protein fused to the translocated promoter remains a limitation. In this study, the c-Abl, c-Src, and c-Met region locus as the result of a gene translocation (ref. 10, and kinases are produced in high yields in Escherichia coli by using a references therein). Further links to cancer have been docu- bicistronic vector encoding the PTP1B tyrosine phosphatase. A mented through the identification of germline mutations in the BIOCHEMISTRY 100-fold lower dose of the inhibitor, Imatinib, was observed to c-Met gene in the majority of hereditary papillary renal carci- inhibit the unphosphorylated form of c-Abl kinase prepared by nomas (11, 12), and in gastric cancer (13). Somatic mutations in using this vector, compared to the phosphorylated form produced the c-Met gene have been identified in sporadic papillary renal without PTP1B, consistent with the known selectivity of this carcinomas (14), small cell lung cancer (15), squamous cell inhibitor for the unactivated conformation of the enzyme. Unphos- cancer of the oropharynx (16), hepatocellular carcinomas (17), phorylated c-Met kinase produced with this vector was used to and lung and lymph node metastases (18, 19). Such truncated obtain the crystal structure, at 2.15-Å resolution, of the autoinhib- and mutated forms of c-Met are found to transform cells in ited form of the kinase domain, revealing an intricate network of culture (18, 20), as well as to cause tumor formation in transgenic interactions involving c-Met residues documented previously to mice (21). When c-Met expression is expressed at high levels in cause dysregulation when mutated in several cancers. mice, it loses its dependence on HGF stimulation (22). However, in the majority of cancers where c-Met plays a role, it is thought autoinhibition ͉ c-Abl ͉ c-Src ͉ cancer to be through a modest induction of c-Met expression levels, and it has been demonstrated that hypoxia can up-regulate the c-Met gene (23–25). Even with activating point mutations, the onco- equencing of the human genome indicates there are Ͼ500 genic actions of c-Met typically still require increased expression different protein kinase genes expressed in man (1). Many of S levels (26, 27), and remain dependent on HGF stimulation (28). these are already known to play important roles in biology, and Strategies to reduce c-Met activity include targeting both the all could potentially be important as targets for pharmaceutical extracellular receptor domain in addition to the intracellular intervention in medicine. Conservation in the active site residues tyrosine kinase domain (23–25, 29–31). within the protein kinase gene family makes the development of The c-Met receptor is composed of an extracellular alpha selective kinase inhibitors challenging. Structural biology offers chain and a transmembrane beta chain, products of a single gene valuable information useful in the design of new inhibitors (2), that become proteolytically cleaved but that remain associated but a limitation in its application to kinases can often be the through a disulfide bond (see ref. 32 for review). Crystal inability to produce highly purified proteins in amounts suitable structures have been reported for the extracellular c-Met Sema for cocrystallography. Inhibitor binding sometimes can be sen- domain (33), as well as a mutated form of the intracellular sitive to the specific conformation state of a kinase (3), or to tyrosine kinase domain (34, 35). Signaling through c-Met is changes in the kinase sequence caused by mutations, such as thought to occur upon HGF binding through dimerization in the those occurring during cancer progression (4–7). These pose membrane (23), leading to activation of the autoinhibited re- further barriers to the implementation of structural approaches ceptor through transphosphorylation. Once phosphorylated, the to drug design, as there can be a need to produce the target intracellular domains intiate a cascade of signaling by binding to kinase in several different forms. several other proteins at a multifunctional docking site linked to Through efforts to create a robust system to produce protein kinases, we discovered that, contrary to common belief, it is possible to produce many kinases in bacteria, including catalytic Conflict of interest statement: W.W., A.M., J.T., A.K., H.I.K., C.Z., B.P., Y.S., H.N., M.T., C.L., domains of receptor tyrosine kinases (8). We discovered that and B.L.W. are shareholders of Plexxikon, Inc., a privately held company. good production systems can be developed by using Escherichia Abbreviation: HGF, hepatocyte growth factor. coli by a simple strategy involving testing many different N- and Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, C-terminal boundaries for optimal expression (8). Such analyses www.pdb.org (PDB ID code 2G15). were previously difficult because of the expense of oligonucle- *To whom correspondence should be addressed. E-mail: [email protected]. otide PCR primers, but these now are readily manageable. We © 2006 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0600048103 PNAS ͉ March 7, 2006 ͉ vol. 103 ͉ no. 10 ͉ 3563–3568 Downloaded by guest on September 29, 2021 Fig. 2. Comparison of dose-dependent inhibition of c-Abl kinase activities by Imatinib. The inhibition of the unphosphorylated (UP) c-Abl produced in E. coli using coexpression with phosphatase occurs with an IC50 of 28 nM (Ϯ5 nM), compared to an IC50 of 3.3 ␮M(Ϯ1.1 ␮M) for the phosphorylated c-Abl produced in E. coli without phosphatase. Points are duplicates normalized to 100% for the uninhibited kinase, with error bars representing the standard deviation of the mean. ities of particular mutations found in patients with hereditary forms of renal cancer. Also in this study an analysis is made of c-Abl and c-Src kinases produced through coexpression with phosphatase in E. coli. Phosphorylation-dependent differences are documented for bacterially expressed c-Abl in the sensitivity to the inhibitor, Imatinib, shown previously to inhibit preferen- tially the unphosphorylated form of c-Abl (3). Thus the use of the phosphatase coexpression system can facilitate the development of kinase inhibitor therapeutics that target different protein conformation states. Results The bicistronic pET-N6 BI-PTP plasmid (Fig. 1A) is a convenient vector for bacterial expression of unphosphorylated ty- Fig. 1. Kinase expression in bacteria. (A) Sequence of the N-terminal HIS-tag rosine kinases. We have engineered several kinases into both this and the NdeI and SalI polylinker region from pET-N6 BI-PTP, the bicistronic vector and the parent pET-N6 vector lacking the PTP (Fig. 1B). expression vector used for coexpression of protein tyrosine kinases with the Thus, c-Abl and c-Src coding sequences were engineered into catalytic fragment of the tyrosine phosphatase, PTP1B. (B) Kinase-encoding these vectors, choosing boundaries similar to ones described in sequences are ligated into vectors without (pET-N6) or with (pET-N6 BI-PTP) earlier structure determination reports. c-Met kinase domain the phosphatase-encoding sequences, for production of the phosphorylated also was engineered into these vectors, but the boundaries had or the unphosphorylated proteins, respectively. (C)(Top) Coomassie-stained SDS͞PAGE of 1 ␮g of the HIS-tagged c-Abl, c-Src, or c-Met kinases after previously been determined empirically by testing several dif- expression without (Ϫ) or with (ϩ) phosphatase coexpression, and after ferent N and C termini for optimal expression (8). Although all purification by metal affinity chromatography. (Middle) Western blot detec- three kinases have previously been produced by using baculo- tion of phospho-Tyr present in 10 ng of the same kinases separated by virus systems, all three were found to express well in E. coli, SDS͞PAGE as in Top.(Bottom) Western blot detection of phospho-Tyr present yielding amounts Ͼ1 mg per liter culture, convenient for making in 10 ␮g of the unpurified soluble protein extract from the E. coli cultures used preparations for crystallography. to produce the kinases in Top and Middle.

Load more