NMR backbone assignments of the tyrosine kinase domain of human fibroblast growth factor receptor 3 in apo state and in complex with inhibitor PD173074

Fibroblast growth factors receptors (FGFR) are transmembrane protein tyrosine kinases involved in many cellular process, including growth, differentiation and angiogenesis. Dysregulation of FGFR enzymatic activity is associated with developmental disorders and cancers; therefore FGFRs have become attractive targets for drug discovery, with a number of agents in late-stage clinical trials. Here, we present the backbone resonance assignments of FGFR3 tyrosine kinase domain in the ligand-free form and in complex with the canonical FGFR kinase inhibitor PD173074. Analysis of chemical shift changes upon inhibitor binding highlights a characteristic pattern of allosteric network perturbations that is of relevance for future drug discovery activities aimed at development of conformationally-selective FGFR inhibitors.     

Clinical Targeting of Mutated and Wild-Type Protein Tyrosine Kinases in Cancer

Clinical therapies for cancer have evolved from toxic, nontargeted agents to manageable, highly targeted therapies. Protein tyrosine kinases are a family of signaling molecules implicated in nearly every cancer type and are the foundation for the development of modern targeted agents. Recent genomic analyses have identified activating mutations, translocations, and amplifications of tyrosine kinases. Selective targeting of these genetically altered tyrosine kinases has resulted in significant clinical advances, including increased patient survival. This indicates that altered protein tyrosine kinases are the main drivers of many different cancers. However, lost during analyses of genetic lesions are the contributions of activated, wild-type kinases on tumor-dependent pathways. New approaches in phosphoproteomic technologies have identified several wild-type tyrosine kinase activation states, suggesting that non-genetically altered kinases can be essential “nodes” for signal transduction. Here, we summarize the evidence supporting the common mechanisms of protein tyrosine kinase activation in cancer and provide a personal perspective on the kinases BCR-ABL and BTK, as well as nonmutated kinase targets in prostate cancer, through our work. We outline the mechanisms of tyrosine kinase activation in the absence of direct mutation and discuss whether non-genetically altered tyrosine kinases or their associated downstream signaling pathways can be effectively targeted.     

Small molecule inhibition of fibroblast growth factor receptors in cancer

Fibroblast growth factors (FGFs) signal through FGF receptors (FGFRs), which are a sub-family of the superfamily of receptor tyrosine kinases, to regulate human development and metabolism. Uncontrolled FGF signaling is responsible for diverse array of developmental disorders, most notably skeletal syndromes due to FGFR gain-of-function mutations. Studies in the last few years have provided significant evidence for the importance of FGF signaling in the pathogenesis of diverse cancers, including endometrial and bladder cancers. FGFs are both potent mitogenic and angiogenic factors and can contribute to carcinogenesis by stimulating cell proliferation and tumor angiogenesis. Gene knockout and pharmacological inhibition of FGFRs in in vivo and in vitro models validate FGFRs as a target for cancer treatment. Considerable efforts are being expended to develop specific, small-molecule inhibitors for treating FGFR-driven cancers. Recent reviews on the FGF/FGFR system have focused primarily on signaling, pathophysiology, and functions in cancer. In this article, we review the key roles of FGFR in cancer, provide an update on the status of clinical trials with small-molecule FGFR inhibitors, and discuss how the current structural data on FGFR kinases guide the design and characterization of new FGFR inhibitors.     

Involvement of protein tyrosine phosphatases in adipogenesis: New anti-obesity targets?

Obesity is a worldwide epidemic as well as being a major risk factor for diabetes, cardiovascular diseases and several types of cancers. Obesity is mainly due to the overgrowth of adipose tissue arising from an imbalance between energy intake and energy expenditure. Adipose tissue, primarily composed of adipocytes, plays a key role in maintaining whole body energy homeostasis. In view of the treatment of obesity and obesity-related diseases, it is critical to understand the detailed signal transduction mechanisms of adipogenic differentiation. Adipogenic differentiation is tightly regulated by many key signal cascades, including insulin signaling. These signal cascades generally transfer or amplify the signal by using serial tyrosine phosphorylations. Thus, protein tyrosine kinases and protein tyrosine phosphatases are closely related to adipogenic differentiation. Compared to protein tyrosine kinases, protein tyrosine phosphatases have received little attention in adipogenic differentiation. This review aims to highlight the involvement of protein tyrosine phosphatases in adipogenic differentiation and the possibility of protein tyrosine phosphatases as drugs to target obesity. [BMB Reports 2012; 45(12): 700-706]     

Novel anticancer drug discovery.

There is at present, much optimism about the possibility of finding selective anticancer drugs that will eliminate the cytotoxic side effects associated with conventional cancer chemotherapy. This hope is based on uncovering many novel molecular targets that are 'cancer-specific', which will allow the targeting of cancer cells while normal cells are spared. Thus far, encouraging results have been obtained with several of these novel agents at the preclinical level, and clinical trials have begun. These targets are involved at one level or more in tumor biology, including tumor cell proliferation, angiogenesis and metastasis. Novel targets for which advances are being made include the following: growth factor receptor tyrosine kinases such as the epidermal growth factor receptor and HER-2/neu (proliferation); the vascular endothelial growth factor receptor and the basic fibroblast growth factor receptor (angiogenesis); the oncogenic GTP-binding protein Ras (especially agents targeting Ras farnesylation, farnesyltransferase inhibitors) (proliferation); protein kinase C (proliferation and drug resistance); cyclin-dependent kinases (proliferation); and matrix metalloproteinases and angiogenin (angiogenesis and metastasis). Less explored, but potentially useful targets include the receptor tyrosine kinase platelet-derived growth factor receptor, mitogen-activated protein kinase cascade oncogenes such as Raf-1 and mitogen-activated protein kinase kinase, cell adhesion molecules such as integrins, anti-apoptosis proteins such as Bcl-2, MDM2 and survivin, and the cell life-span target telomerase.     

Abl kinases are required for invadopodia formation and chemokine-induced invasion

The Abl tyrosine kinases, Abl and Arg, play a role in the regulation of the actin cytoskeleton by modulating cell-cell adhesion and cell motility. Deregulation of both the actin cytoskeleton and Abl kinases have been implicated in cancers. Abl kinase activity is elevated in a number of metastatic cancers and these kinases are activated downstream of several oncogenic growth factor receptor signaling pathways. However, the role of Abl kinases in regulation of the actin cytoskeleton during tumor progression and invasion remains elusive. Here we identify the Abl kinases as essential regulators of invadopodia assembly and function. We show that Abl kinases are activated downstream of the chemokine receptor, CXCR4, and are required for cancer cell invasion and matrix degradation induced by SDF1α, serum growth factors, and activated Src kinase. Moreover, Abl kinases are readily detected at invadopodia assembly sites and their inhibition prevents the assembly of actin and cortactin into organized invadopodia structures. We show that active Abl kinases form complexes with membrane type-1 matrix metalloproteinase (MT1-MMP), a critical invadopodia component required for matrix degradation. Further, loss of Abl kinase signaling induces internalization of MT1-MMP from the cell surface, promotes its accumulation in the perinuclear compartment and inhibits MT1-MMP tyrosine phosphorylation. Our findings reveal that Abl kinase signaling plays a critical role in invadopodia formation and function, and have far-reaching implications for the treatment of metastatic carcinomas.     

Involvement of JAK-family tyrosine kinases in hematopoietin receptor signal transduction

A variety of cytokines, hormones and hematopoietic growth factors signal through the hematopoietin family of membrane receptors, which share several structural features, including a Trp-Ser-X-Trp-Ser motif and four paired cysteine residues. The signal transduction mechanisms utilized by these receptors have remained elusive, although tyrosine kinase activation has been one common element. Recently, a role for the cytoplasmic tyrosine kinases of the Janus kinase (JAK) family has been implicated in signalling by these receptors. There are currently three known JAK family kinases, including JAK1, JAK2 and TYK2. This review will focus on the role of such tyrosine kinases in hematopoietin receptor signal transduction, and address the possibility of the involvement also of unidentified Janus kinases.     

A colorimetric enzyme-linked on-bead assay for identification of synthetic substrates of protein tyrosine kinases.

Protein tyrosine kinases play key roles in the progression of numerous human diseases including several types of cancers. We report here a simple colorimetric assay for tyrosine kinase activity employing synthetic peptide substrates prepared on Tentagel synthesis beads. Phosphorylation of compounds on beads was detected with an antiphosphotyrosine antibody complexed with a secondary antibody-alkaline phosphatase conjugate. This assay may prove useful for the identification and characterization of synthetic substrates of this important class of enzymes.     

Tyrosine kinase mutations in human cancer

A subset of tyrosine kinases are activated by mutations which contribute to the malignant transformation, growth, and metastasis of human cancers. Mutations change the expression, conformation and/or stability of tyrosine kinases, often leading to constitutive activation of the signaling pathways the kinases regulate. Given that tyrosine kinases are key members of signaling cascades, mutations have multiple effects on various cellular proteins. This review will focus on four kinases (EGFR, c-Met, c-Kit, and PI3-kinase) known to be mutated in human cancer. It will discuss the effects that these mutations have on the biology of tumors, and how our understanding of the structure and function of kinases and their mutations is currently being used to design targeted treatments.     

Evolution of Functional Diversity in the Holozoan Tyrosine Kinome

The emergence of multicellularity is strongly correlated with the expansion of tyrosine kinases, a conserved family of signaling enzymes that regulates pathways essential for cell-to-cell communication. Although tyrosine kinases have been classified from several model organisms, a molecular-level understanding of tyrosine kinase evolution across all holozoans is currently lacking. Using a hierarchical sequence constraint-based classification of diverse holozoan tyrosine kinases, we construct a new phylogenetic tree that identifies two ancient clades of cytoplasmic and receptor tyrosine kinases separated by the presence of an extended insert segment in the kinase domain connecting the D and E-helices. Present in nearly all receptor tyrosine kinases, this fast-evolving insertion imparts diverse functionalities, such as post-translational modification sites and regulatory interactions. Eph and EGFR receptor tyrosine kinases are two exceptions which lack this insert, each forming an independent lineage characterized by unique functional features. We also identify common constraints shared across multiple tyrosine kinase families which warrant the designation of three new subgroups: Src module (SrcM), insulin receptor kinase-like (IRKL), and fibroblast, platelet-derived, vascular, and growth factor receptors (FPVR). Subgroup-specific constraints reflect shared autoinhibitory interactions involved in kinase conformational regulation. Conservation analyses describe how diverse tyrosine kinase signaling functions arose through the addition of family-specific motifs upon subgroup-specific features and coevolving protein domains. We propose the oldest tyrosine kinases, IRKL, SrcM, and Csk, originated from unicellular premetazoans and were coopted for complex multicellular functions. The increased frequency of oncogenic variants in more recent tyrosine kinases suggests that lineage-specific functionalities are selectively altered in human cancers.     

The Achilles Heel of ErbB-2/HER2: Regulation by the Hsp90 Chaperone Machine and Potential for Pharmacological Intervention

Signal transduction mediated by ErbB/HER receptor tyrosine kinases is crucial for the development and maintenance of epithelial tissues, and aberrant signaling is frequently associated with malignancies of epithelial origin. This review focuses on the roles played by the Hsp90 chaperone machinery in the regulation of signaling through the ErbB/HER network, and discusses potential therapeutic strategies that disrupt chaperone functions. Hsp90 and its associated co-chaperones regulate ErbB signal transduction through multiple mechanisms. The chaperone system controls the stability of the nascent forms of both ErbB-1 (EGF-receptor) and ErbB-2/HER2, while regulation of the mature form is restricted to ErbB-2. Regulation by the Hsp90 complex extends to downstream effectors of ErbB signaling, namely Raf-1, Pdk-1 and Akt/PKB. Disrupting the function of Hsp90 results in the degradation of both the receptors and their effectors, thereby inhibiting tumor cell growth. The importance of an Hsp90-recognition motif located within the kinase domain of ErbB-2 is discussed, as well as a direct role for Hsp90 in regulating tyrosine kinase activity. In light of recent observations, we emphasize the ability of specific tyrosine kinase inhibitors to selectively target ErbB-2 to the chaperone-mediated degradation pathway. ErbB-specific drugs are already used to treat cancers, and clinical trials are underway for additional compounds that intercept ErbB signaling, including drugs that target Hsp90. Hence, the dependence of ErbB-2 upon Hsp90 reveals an Achilles heel, which opens a window of opportunity for combating cancers driven by the ErbB/HER signaling network.     

The achilles heel of ErbB-2/HER2: regulation by the Hsp90 chaperone machine and potential for pharmacological intervention

Signal transduction mediated by ErbB/HER receptor tyrosine kinases is crucial for the development and maintenance of epithelial tissues, and aberrant signaling is frequently associated with malignancies of epithelial origin. This review focuses on the roles played by the Hsp90 chaperone machinery in the regulation of signaling through the ErbB/HER network, and discusses potential therapeutic strategies that disrupt chaperone functions. Hsp90 and its associated cochaperones regulate ErbB signal transduction through multiple mechanisms. The chaperone system controls the stability of the nascent forms of both ErbB-1 (EGF-receptor) and ErbB-2/HER2, while regulation of the mature form is restricted to ErbB-2. Regulation by the Hsp90 complex extends to downstream effectors of ErbB signaling, namely Raf-1, Pdk-1 and Akt/PKB. Disrupting the function of Hsp90 results in the degradation of both the receptors and their effectors, thereby inhibiting tumor cell growth. The importance of an Hsp90-recognition motif located within the kinase domain of ErbB-2 is discussed, as well as a direct role for Hsp90 in regulating tyrosine kinase activity. In light of recent observations, we emphasize the ability of specific tyrosine kinase inhibitors to selectively target ErbB-2 to the chaperone-mediated degradation pathway. ErbB-specific drugs are already used to treat cancers, and clinical trials are underway for additional compounds that intercept ErbB signaling, including drugs that target Hsp90. Hence, the dependence of ErbB-2 upon Hsp90 reveals an Achilles heel, which opens a window of opportunity for combating cancers driven by the ErbB/HER signaling network.     

Tyrosine kinase signaling in fibrotic disorders: Translation of basic research to human disease

Tyrosine kinases regulate a broad variety of physiological cell processes, including metabolism, growth, differentiation and apoptosis. Abnormal tyrosine kinase activity disturbs the physiological cell homeostasis and can lead to cancer, vascular disease, and fibrosis. In regard to fibrosis, different tyrosine kinases have been identified as determinants of disease progression and potential targets for anti-fibrotic therapies. This includes both receptor tyrosine kinases (e.g., PDGF receptor, VEGF receptor, EGF receptor, and JAK kinases) as well as non-receptor tyrosine kinases (e.g., c-Abl, c-Kit, and Src kinases). Given their central role in the pathogenesis of fibrosis, researchers of our field study the anti-fibrotic effects of monoclonal antibodies or small-molecule inhibitors to block the aberrant tyrosine kinase activity and treat fibrosis in preclinical models of various fibrotic diseases (e.g., idiopathic pulmonary fibrosis, renal fibrosis, liver fibrosis, and dermal fibrosis). The results of these studies were promising and prompted clinical trials with different compounds in fibrotic diseases. So far, results from studies with intedanib in idiopathic pulmonary fibrosis and imatinib in idiopathic pulmonary fibrosis and systemic sclerosis have been reported. Although none of these studies reported a positive primary outcome, promising trends in anti-fibrotic efficacy awaken our hopes for a new class of effective anti-fibrotic targeted therapies. This article is part of a Special Issue entitled: Fibrosis: Translation of basic research to human disease.     

EGFR and c-Met Cross Talk in Glioblastoma and Its Regulation by Human Cord Blood Stem Cells

Receptor tyrosine kinases (RTK) and their ligands control critical biologic processes, such as cell proliferation, migration, and differentiation. Aberrant expression of these receptor kinases in tumor cells alters multiple downstream signaling cascades that ultimately drive the malignant phenotype by enhancing tumor cell proliferation, invasion, metastasis, and angiogenesis. As observed in human glioblastoma (hGBM) and other cancers, this dysregulation of RTK networks correlates with poor patient survival. Epidermal growth factor receptor (EGFR) and c-Met, two well-known receptor kinases, are coexpressed in multiple cancers including hGBM, corroborating that their downstream signaling pathways enhance a malignant phenotype. The integration of c-Met and EGFR signaling in cancer cells indicates that treatment regimens designed to target both receptor pathways simultaneously could prove effective, though resistance to tyrosine kinase inhibitors continues to be a substantial obstacle. In the present study, we analyzed the antitumor efficacy of EGFR inhibitors erlotinib and gefitinib and c-Met inhibitor PHA-665752, along with their respective small hairpin RNAs (shRNAs) alone or in combination with human umbilical cord blood stem cells (hUCBSCs), in glioma cell lines and in animal xenograft models. We also measured the effect of dual inhibition of EGFR/c-Met pathways on invasion and wound healing. Combination treatments of hUCBSC with tyrosine kinase inhibitors significantly inhibited invasion and wound healing in U251 and 5310 cell lines, thereby indicating the role of hUCBSC in inhibition of RTK-driven cell behavior. Further, the EGFR and c-Met localization in glioma cells and hGBM clinical specimens indicated that a possible cross talk exists between EGFR and c-Met signaling pathway.     

The EphB4 Receptor Tyrosine Kinase Promotes Lung Cancer Growth: A Potential Novel Therapeutic Target

Despite progress in locoregional and systemic therapies, patient survival from lung cancer remains a challenge. Receptor tyrosine kinases are frequently implicated in lung cancer pathogenesis, and some tyrosine kinase inhibition strategies have been effective clinically. The EphB4 receptor tyrosine kinase has recently emerged as a potential target in several other cancers. We sought to systematically study the role of EphB4 in lung cancer. Here, we demonstrate that EphB4 is overexpressed 3-fold in lung tumors compared to paired normal tissues and frequently exhibits gene copy number increases in lung cancer. We also show that overexpression of EphB4 promotes cellular proliferation, colony formation, and motility, while EphB4 inhibition reduces cellular viability in vitro, halts the growth of established tumors in mouse xenograft models when used as a single-target strategy, and causes near-complete regression of established tumors when used in combination with paclitaxel. Taken together, these data suggest an important role for EphB4 as a potential novel therapeutic target in lung cancer. Clinical trials investigating the efficacy of anti-EphB4 therapies as well as combination therapy involving EphB4 inhibition may be warranted.     

Structure and function of tyrosine kinase receptors

Over the past ten years, several growth factor receptors have been shown to be ligand-regulated tyrosine kinases. Tyrosine kinase activity is essential for signal transmission, suggesting that phosphorylation cascades may play an important role. Considerable effort has gone into understanding the structure and function of tyrosine kinase receptors in order to define their mechanisms of signal transmission. However, the protein substrates of the receptor kinases have proven to be difficult to isolate and clone. This review focuses on the receptors for insulin, epidermal growth factor, and platelet-derived growth factor. They are all tyrosine kinases, but emerging evidence suggests that they utilize multiple separate signal transduction pathways. Work carried out during the next several years should yield considerable insight into the complexity of the components which interact with these tyrosine kinase receptors to regulate cellular growth and metabolism.     

Protein kinases 1988: a current perspective

This review focuses on several recent developments in the field of protein kinases. In the area of protein serine/threonine kinases, much has been learned recently about protein kinase C structure and function. Novel lipid mediators, both stimulatory and inhibitory, have been discovered, and kinase has been shown to be an increasingly large family of gene products. Heterogeneity of cellular localization and function has been documented. Calcium/calmodulin-dependent protein kinases are now believed to consist of at least five enzymes, which range from those with extreme substrate specificity such as phosphorylase kinase and myosin light-chain kinases to calcium calmodulin kinase II, with several known substrates. Several of these enzymes appear to be important in synaptic transmission and, for calcium/calmodulin kinase III, in the regulation of protein synthesis. Several new examples of pseudosubstrate prototopes as endogenous kinase inhibitors have been described, including regions intrinsic to kinase primary sequences, which could serve as constitutive inhibitors of enzyme activity. In the field of protein tyrosine kinases, new enzyme species are being discovered at a rapid rate. There are several well-documented examples of kinase autophosphorylation on tyrosine leading to stimulation of catalytic activity. For the growth factor receptors with intrinsic protein tyrosine kinase activity, it now seems clear that kinase catalytic activity is necessary for most hormone effects on cells, with the general exceptions of ligand binding and, possibly, receptor cycling. Finally, several groups have recently described a close association between protein tyrosine kinases and a phosphatidylinositol kinase activity, a link that might eventually explain some of the initial steps in signal transduction that occur after kinase activation.     

Heme controls the regulation of protein tyrosine kinases Jak2 and Src

Protein tyrosine kinases play key roles in many molecular and cellular processes in diverse living organisms. Their proper functioning is crucial for the normal growth, development, and health in humans, whereas their dysfunction can cause serious diseases, including various cancers. As such, intense studies have been performed to understand the molecular mechanisms by which the activities of protein tyrosine kinases are regulated in mammalian cells. Particularly, small molecules that can modulate the activity of tyrosine kinases are of great importance for discovering therapeutic drug candidates for numerous diseases. Notably, heme cannot only serve as a prosthetic group for hemoglobins and enzymes, but it also is a small signaling molecule that can control the activity of diverse signaling and regulatory proteins. Using a computational search, we found that a group of non-membrane spanning tyrosine kinases contains one or more CP motifs that can potentially bind to heme and mediate heme regulation. We then used experimental approaches to determine whether heme can affect the activity of any of these tyrosine kinases. We found that heme indeed affects the phosphorylation of key tyrosine residues in Jak2 and Src, and is therefore able to modulate Jak2 and Src activity. Further experiments showed that Jak2 and Src bind to heme and that the presence of heme alters the sensitivity of Jak2 and Src to trypsin digestion. These results suggest that heme actively interacts with Jak2 and Src and alters their conformation.     

Tyrosine phosphatases as a superfamily of tumor suppressors in colorectal cancer

Phosphorylation and dephosphorylation processes catalyzed by numerous kinases and phosphorylases are essential for cell homeostasis and may lead to disturbances in a variety of vital cellular pathways, such as cell proliferation and differentiation, and thus to complex diseases including cancer. As over 80 % of all oncogenes encode protein tyrosine kinases (PTKs), protein tyrosine phosphatases (PTPs), which can reverse the effects of tyrosine kinases, are very important tumor suppressors. Alterations in tyrosine kinase and phosphatase genes including point mutations, changes in epigenetic regulation, as well as chromosomal aberrations involving regions critical to these genes, are frequently observed in a variety of cancers. Colorectal cancer (CRC) is one of the most common cancers in humans. CRCs occur in a familial (about 15 % of all cases), hereditary (about 5%) and sporadic (almost 75-80 %) form. As genetic-environmental interrelations play an important role in the susceptibility to sporadic forms of CRCs, many studies are focused on genetic alterations in such tumors. Mutational analysis of the tyrosine phosphatome in CRCs has identified somatic mutations in PTPRG, PTPRT, PTPN3, PTPN13 and PTPN14. The majority of these mutations result in a loss of protein function. Also, alterations in the expression of these genes, such as decreased expression of PTPRR, PTPRO, PTPRG and PTPRD, mediated by epigenetic mechanisms have been observed in a variety of tumors. Since cancer is a social and global problem, there will be a growing number of studies on alterations in the candidate cancer genes, including protein kinases and phosphatases, to determine the origin, biology and potential pathways for targeted anticancer therapy.