CD2AP/CIN85 balance determines receptor tyrosine kinase signaling response in podocytes

Defects in podocyte signaling are the basis of many inherited glomerular diseases leading to glomerulosclerosis. CD2-associated protein (CD2AP) is highly expressed in podocytes and is considered to play an important role in the maintenance of the glomerular slit diaphragm. Mice deficient for CD2AP (CD2AP(-/-)) appear normal at birth but develop a rapid onset nephrotic syndrome at 3 weeks of age. We demonstrate that impaired intracellular signaling with subsequent podocyte damage is the reason for this delayed podocyte injury in CD2AP(-/-) mice. We document that CD2AP deficiency in podocytes leads to diminished signal initiation and termination of signaling pathways mediated by receptor tyrosine kinases (RTKs). In addition, we demonstrate that CIN85, a paralog of CD2AP, is involved in termination of RTK signaling in podocytes. CIN85 protein expression is increased in CD2AP(-/-) podocytes in vitro. Stimulation of CD2AP(-/-) podocytes with various growth factors, including insulin-like growth factor 1, vascular endothelial growth factor, and fibroblast growth factor, resulted in a significantly decreased phosphatidylinositol 3-kinase/AKT and ERK signaling response. Moreover, increased CIN85 protein is detectable in podocytes in diseased CD2AP(-/-) mice, leading to decreased base-line activation of ERK and decreased phosphorylation after growth factor stimulation in vivo. Because repression of CIN85 protein leads to a restored RTK signaling response, our results support an important role of CD2AP/CIN85 protein balance in the normal signaling response of podocytes.     

Conjugation to Nedd8 instigates ubiquitylation and down-regulation of activated receptor tyrosine kinases

When appended to the epidermal growth factor receptor (EGFR), ubiquitin serves as a sorting signal for lysosomal degradation. Here we demonstrate that the ubiquitin ligase of EGFR, namely c-Cbl, also mediates receptor modification with the ubiquitin-like molecule Nedd8. EGF stimulates receptor neddylation, which enhances subsequent ubiquitylation, as well as sorting of EGFR for degradation. Multiple lysine residues, located within the tyrosine kinase domain of EGFR, serve as attachment sites for Nedd8. A set of clathrin coat-associated binders of ubiquitin also bind Nedd8, but they undergo ubiquitylation, not neddylation. We discuss the emerging versatility of the concerted action of ubiquitylation and neddylation in the process that desensitizes growth factor-activated receptor tyrosine kinases.     

Transglutaminases and receptor tyrosine kinases

Transglutaminases are confounding enzymes which are known to play key roles in various cellular processes. In this paper, we aim to bring together several pieces of evidence from published research and literature that suggest a potentially vital role for transglutaminases in receptor tyrosine kinases (RTK) signalling. We cite literature that confirms and suggests the formation of integrin:RTK:transglutaminase complexes and explores the occurrence and functionality of these complexes in a large fraction of the RTK family.     

Dab2 links CIN85 with clathrin-mediated receptor internalization

CIN85 is a multidomain scaffold protein involved in downregulation of receptor tyrosine kinases. Here we show that disabled-2 (Dab2), an endocytic adaptor molecule implicated in clathrin-coat assembly, associates with CIN85 in mammalian cells. All three SH3 domains of CIN85 were able to bind to the PKPAPR peptide in the carboxyl-terminal part of Dab2, possibly enabling CIN85 to simultaneously interact with multiple Dab2 molecules. CIN85 association with Dab2 is essential for its recruitment to clathrin coat and appears to be modulated by growth factor stimulation. Dab2 and clathrin dissociated from CIN85 following growth factor treatment, enabling other molecules, such as Cbl, to bind to CIN85. Taken together, our data indicate a dynamic interplay between CIN85 and its effectors during endocytosis of receptor tyrosine kinases.     

Structural analysis of receptor tyrosine kinases

Receptor tyrosine kinases (RTKs) are single-pass transmembrane receptors that possess intrinsic cytoplasmic enzymatic activity, catalyzing the transfer of the γ-phosphate of ATP to tyrosine residues in protein substrates. RTKs are essential components of signal transduction pathways that affect cell proliferation, differentiation, migration and metabolism. Included in this large protein family are the insulin receptor and the receptors for growth factors such as epidermal growth factor, fibroblast growth factor and vascular endothelial growth factor. Receptor activation occurs through ligand binding, which facilitates receptor dimerization and autophosphorylation of specific tyrosine residues in the cytoplasmic portion. The phosphotyrosine residues either enhance receptor catalytic activity or provide docking sites for downstream signaling proteins. Over the past several years, structural studies employing X-ray crystallography have advanced our understanding of the molecular mechanisms by which RTKs recognize their ligands and are activated by dimerization and tyrosine autophosphorylation. This review will highlight the key results that have emerged from these structural studies.     

Antibodies directed against receptor tyrosine kinases

Approximately 30 therapeutic monoclonal antibodies have already been approved for cancers and inflammatory diseases, and monoclonal antibodies continue to be one of the fastest growing classes of therapeutic molecules. Because aberrant signaling by receptor tyrosine kinases (RTKs) is a commonly observed factor in cancer, most of the subclasses of RTKs are being extensively studied as potential targets for treating malignancies. The first two RTKs that have been targeted by antibody therapy, with five currently marketed antibodies, are the growth factor receptors EGFR and HER2. However, due to systemic side effects, refractory patients and the development of drug resistance, these treatments are being challenged by emerging therapeutics. This review examines current monoclonal antibody therapies against RTKs. After an analysis of agents that have already been approved, we present an analysis of antibodies in clinical development that target RTKs. Finally, we highlight promising RTKs that are emerging as new oncological targets for antibody-based therapy.     

Endothelial receptor tyrosine kinases involved in angiogenesis

The Saccharomyces cerevisiae EMP47 gene encodes a nonessential type-I transmembrane protein with sequence homology to a class of intracellular lectins defined by ERGIC-53 and VIP36. The 12-amino acid COOH-terminal cytoplasmic tail of Emp47p ends in the sequence KTKLL, which conforms with the consensus for di-lysine-based ER-localization signals. Despite the presence of this motif, Emp47p was shown to be a Golgi protein at steady-state. The di-lysine motif of Emp47p was functional when transplanted onto Ste2p, a plasma membrane protein, conferring ER localization. Nevertheless, the di-lysine motif was required for Golgi-localization of Emp47p and showed the same charge- independent, position-dependent characteristics of other di-lysine motifs. Alpha-COP has been shown to be required for ER localization of di-lysine-tagged proteins. Consistent with this finding, the Ste2p- Emp47p hybrid protein was mislocalized to the cell surface in the alpha- COP mutant, ret1-1. Surprisingly, the Golgi-localization of Emp47p was unaffected by the ret1-1 mutation. To investigate whether Emp47p undergoes retrograde transport from the Golgi to the ER like other di- lysine-tagged proteins we developed an assay to measure this step after block of forward transport in a sec12 mutant. Under these conditions retrograde transport led to a specific redistribution of Emp47p from the Golgi to the ER. This recycling occurred from a Golgi subcompartment containing alpha 1,3 mannose-modified oligosaccharides suggesting that it originated from a medial-or later Golgi compartment. Thus Emp47p cycles between the Golgi apparatus and the ER and requires a di-lysine motif for its alpha-COP-independent, steady state localization in the Golgi.     

CIN85 regulates dopamine receptor endocytosis and governs behaviour in mice

Despite extensive investigations of Cbl‐interacting protein of 85 kDa (CIN85) in receptor trafficking and cytoskeletal dynamics, little is known about its functions in vivo. Here, we report the study of a mouse deficient of the two CIN85 isoforms expressed in the central nervous system, exposing a function of CIN85 in dopamine receptor endocytosis. Mice lacking CIN85 exon 2 (CIN85^Δex2) show hyperactivity phenotypes, characterized by increased physical activity and exploratory behaviour. Interestingly, CIN85^Δex2 animals display abnormally high levels of dopamine and D2 dopamine receptors (D2DRs) in the striatum, an important centre for the coordination of animal behaviour. Importantly, CIN85 localizes to the post‐synaptic compartment of striatal neurons in which it co‐clusters with D2DRs. Moreover, it interacts with endocytic regulators such as dynamin and endophilins in the striatum. Absence of striatal CIN85 causes insufficient complex formation of endophilins with D2DRs in the striatum and ultimately decreased D2DR endocytosis in striatal neurons in response to dopamine stimulation. These findings indicate an important function of CIN85 in the regulation of dopamine receptor functions and provide a molecular explanation for the hyperactive behaviour of CIN85^Δex2 mice.     

Defective downregulation of receptor tyrosine kinases in cancer

Most growth factors control cellular functions by activating specific receptor tyrosine kinases (RTKs). While overactivation of RTK signalling pathways is strongly associated with carcinogenesis, it is becoming increasingly clear that impaired deactivation of RTKs may also be a mechanism in cancer. A major deactivation pathway, receptor downregulation, involves ligand-induced endocytosis of the RTK and subsequent degradation in lysosomes. A complex molecular machinery that uses the small protein ubiquitin as a key regulator assures proper endocytosis and degradation of RTKs. Here we discuss evidence that implicates deregulation of this machinery in cancer.     

Leucine motif-dependent tyrosine autophosphorylation of type III receptor tyrosine kinases

Activation loop tyrosine autophosphorylation is an essential requirement for full kinase activation of receptor tyrosine kinases (RTKs). However, mechanisms involved are not fully understood. In general, kinase domains of RTKs are folded into two main lobes, NH2- and COOH-terminal lobes. The COOH-terminal lobe of vascular endothelial growth factor receptor-2 (VEGFR-2) is folded into seven alpha-helices (alphaD-alphaI). In the studies presented here we demonstrate that leucine residues of helix I (alphaI) regulate tyrosine autophosphorylation and phosphotransferase activity of VEGFR-2. The presence of leucines 1158, 1161, and 1162 are essential for tyrosine autophosphorylation and kinase activation of VEGFR-2 and are involved in helix-helix packing via hydrophobic interactions. The presence of leucine 1158 is critical for kinase activation of VEGFR-2 and appears to interact with alphaE, alphaF, alphaH, and beta7. The analogous residue, leucine 957 on platelet-derived growth factor receptor-beta and leucine 910 on colony stimulating factor-1R are also found to be critical for tyrosine autophosphorylation of these receptors. Leucines 1161 and 1162 are also involved in helix-helix packing but they play a less critical role in VEGFR-2 activation. Thus, we conclude that leucine motif-mediated helix-helix interactions are critical for kinase regulation of type III RTKs. This mechanism is likely to be shared with other kinases and might provide a basis for the design of a novel class of tyrosine kinase inhibitors.     

Characterization of the CIN85 adaptor protein and identification of components involved in CIN85 complexes

CIN85 is an 85-kDa adaptor protein whose functions in signaling pathways are presently unknown. Using the yeast two-hybrid screen, the B cell linker protein (BLNK) was identified as a binding partner of CIN85. Coimmunoprecipitation experiments using mammalian cells revealed that CIN85 directly bound to BLNK through its SH3 domains. Immunostaining analysis showed that CIN85 and BLNK were colocalized in the cytoplasm. These results indicate a potential role of CIN85 in the B cell receptor-mediated signaling pathway. It was also found that Crk-I, Crk-II, p130(Cas), p85-PI3K, Grb2, and Sos1 were components of CIN85 complexes. CIN85 interacted with itself through its coiled-coil region, resulting in formation of a tetramer. Both the coiled-coil region and SH3 domains of CIN85 were responsible for its subcellular localization. Our data suggest that CIN85 may serve for regulation of various signaling events through formation of its diverse complexes.     

Compartmentalized signal transduction by receptor tyrosine kinases

Signal transduction through receptor tyrosine kinases is believed to occur mainly at the plasma membrane. Ligands bind to their cognate receptors and trigger autophosphorylation events, which are detected by intracellular signalling molecules. However, ligands, such as epidermal growth factor and insulin, induce the rapid internalization of their receptors into endosomes. Although this event is traditionally thought to attenuate the ligand-induced response, in this article the authors discuss an alternative scenario in which selective and regulated signal transduction from receptor tyrosine kinases occurs within the endosome.     

Roles of receptor tyrosine kinases in Drosophila development.

Communication between cells is a fundamental component of development and morphogenesis. Identification of the molecules mediating cell-cell communication is crucial for elucidation of the molecular basis of these processes. Receptor tyrosine kinases (RTKs) appear to play a central role in this context by transmitting into cells information dictating their fate. The functions of RTKs in Drosophila are extremely diverse, and include maternal determination of embryonic polarity (torso and torpedo), determination of neuroblast identity (faint little ball), and guidance of tracheal cell migration in the embryo (breathless). During compound eye development, RTKs affect the number of photoreceptor clusters (Ellipse) and the determination of photoreceptor R7 identity (sevenless). The phenotypes of mutations in RTK loci serve as a starting point for understanding processes dictating cell identity at the level of the whole organism. Recently, they have also begun to provide a basis for selection of second-site suppressor mutations, encoding additional elements in their signal transduction pathway. Common themes between the functions, regulation, and signal transduction pathways of Drosophila RTKs are drawn.     

Localization and modulation of ErbB receptor tyrosine kinases

The ErbB family of receptor tyrosine kinases serves as a model for understanding the propagation of growth factor signals across the plasma membrane and the interpretation of those signals into a cellular response. Recent studies point to a critical role for the accumulation of ErbBs at specific cell-surface locations in the fidelity of ErbB signaling. The past year has witnessed significant advances in our understanding of the molecular mechanisms of ErbB localization and the role of PDZ-domain-containing proteins and cell-surface glycoproteins in directly modulating signaling through ErbBs.     

Mechanism of Csk-mediated down-regulation of Src family tyrosine kinases in epidermal growth factor signaling

The Src family tyrosine kinases (SFKs) play pivotal roles as molecular switches that link a variety of extracellular cues to intracellular signaling pathway. The function of SFK is regulated by phosphorylation at the C-terminal regulatory site mediated by Csk. Recently a novel SFK target Cbp (or PAG) was identified as a membrane-anchored scaffold protein for Csk. To establish the mechanism of Csk/Cbp-mediated regulation of SFK in vivo, we observed dynamic changes in the interaction of Csk with Cbp by utilizing fusion proteins with modified green fluorescent proteins: cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP). Upon SFK activation induced by epidermal growth factor stimulation, fluorescent resonance energy transfer (FRET) response was detected transiently at membrane ruffles in COS1 cells co-expressing CFP-Csk and Cbp-YFP and in cells expressing a single-molecule FRET indicator consisting of CskSH2 and Cbp. Suppression of SFK by PP2 or use of a mutant Cbp that lacks the Csk binding site abolished the FRET response, although a dominant-negative form of Csk enhanced and sustained the FRET response, demonstrating that the FRET response is dependent upon the SFK activity. These observations show that Csk/Cbp-mediated down-regulation of SFK takes place at membrane ruffles in an early stage of epidermal growth factor signaling and suggest that the Csk/Cbp-based FRET indicators are useful for monitoring the status of SFK in living cells.     

Inhibitors of angiogenesis and cancer-related receptor tyrosine kinases

Inhibition of tumor angiogenesis is an attractive target in cancer therapy. In this context, receptor tyrosine kinases play a pivotal role. Extensive efforts have been made to identify and develop small-molecule inhibitors of these central signaling proteins. Some of these compounds have already passed or are currently in clinical trials to investigate their applicability as anti-cancer drugs. However, the high expectations that are set in antiangiogenic therapy have not yet been accomplished. But there are also new and exciting opportunities for cancer treatment by combining antiangiogenic molecules with newly emerging therapeutics.