Catalytic domains of tyrosine kinases determine the phosphorylation sites within c-Cbl

Catalytic (SH1) domains of protein tyrosine kinases (PTKs) demonstrate specificity for peptide substrates. Whether SH1 domains differentiate between tyrosines in a physiological substrate has not been confirmed. Using purified proteins, we studied the ability of Syk, Fyn, and Abl to differentiate between tyrosines in a common PTK substrate, c-Cbl. We found that each kinase produced a distinct pattern of c-Cbl phosphorylation, which altered the phosphotyrosine-dependent interactions between c-Cbl and CrkL or phosphatidylinositol 3'-kinase (PI3-K). Our data support the concept that SH1 domains determine the final sites of phosphorylation once PTKs reach their target proteins.     

Origin and molecular evolution of receptor tyrosine kinases with immunoglobulin-like domains

Receptor tyrosine kinases (RTKs) are involved in the control of fundamental cellular processes in metazoans. In vertebrates, RTK could be grouped in distinct classes based on the nature of their cognate ligand and modular composition of their extracellular domain. RTK with immunoglobulin-like domains (IG-like RTK) encompass several RTK classes and have been found in early metazoans, including sponges. Evolution of IG-like RTK is characterized by extended molecular and functional diversification, which prompted us to study their evolutionary history. For that purpose, a nonredundant data set including annotated protein sequences of IG-like RTK (n = 85) was built, representing 19 species ranging from sponges to humans. Phylogenetic trees were generated from alignment of conserved regions using maximum likelihood approach. Molecular phylogeny strongly suggests that IG-like RTK diversification occurred according to a complex scenario. In particular, we propose that specific cis duplications of a common ancestor to both platelet-derived growth factor receptor (class III) and vascular endothelial growth factor receptor (class V) families preceded two trans duplications. In contrast, other IG-like RTK genes, like Musk and PTK7, apparently did not evolve by duplications, whereas fibroblast growth factor receptors (class IV) evolved through two rounds of trans duplications. The proposed model of IG-like RTK evolution is supported by high bootstrap values and by the clustering of genes encoding class III and class V RTKs at specific chromosomal locations in mouse and human genomes.     

Functions of the ectodomain and cytoplasmic tyrosine phosphatase domains of receptor protein tyrosine phosphatase Dlar in vivo

The receptor protein tyrosine phosphatase (PTPase) Dlar has an ectodomain consisting of three immunoglobulin (Ig)-like domains and nine fibronectin type III (FnIII) repeats and a cytoplasmic domain consisting of two PTPase domains, membrane-proximal PTP-D1 and C-terminal PTP-D2. A series of mutant Dlar transgenes were introduced into the Drosophila genome via P-element transformation and were then assayed for their capacity to rescue phenotypes caused by homozygous loss-of-function genotypes. The Ig-like domains, but not the FnIII domains, are essential for survival. Conversely, the FnIII domains, but not the Ig-like domains, are required during oogenesis, suggesting that different domains of the Dlar ectodomain are involved in distinct functions during Drosophila development. All detectable PTPase activity maps to PTP-D1 in vitro. The catalytically inactive mutants of Dlar were able to rescue Dlar(-/-) lethality nearly as efficiently as wild-type Dlar transgenes, while this ability was impaired in the PTP-D2 deletion mutants DlarDeltaPTP-D2 and Dlar(bypass). Dlar-C1929S, in which PTP-D2 has been inactivated, increases the frequency of bypass phenotype observed in Dlar(-/-) genotypes, but only if PTP-D1 is catalytically active in the transgene. These results indicate multiple roles for PTP-D2, perhaps by acting as a docking domain for downstream elements and as a regulator of PTP-D1.     

The Src module: an ancient scaffold in the evolution of cytoplasmic tyrosine kinases

Tyrosine kinases were first discovered as the protein products of viral oncogenes. We now know that this large family of metazoan enzymes includes nearly one hundred structurally diverse members. Tyrosine kinases are broadly classified into two groups: the transmembrane receptor tyrosine kinases, which sense extracellular stimuli, and the cytoplasmic tyrosine kinases, which contain modular ligand-binding domains and propagate intracellular signals. Several families of cytoplasmic tyrosine kinases have in common a core architecture, the “Src module,” composed of a Src-homology 3 (SH3) domain, a Src-homology 2 (SH2) domain, and a kinase domain. Each of these families is defined by additional elaborations on this core architecture. Structural, functional, and evolutionary studies have revealed a unifying set of principles underlying the activity and regulation of tyrosine kinases built on the Src module. The discovery of these conserved properties has shaped our knowledge of the workings of protein kinases in general, and it has had important implications for our understanding of kinase dysregulation in disease and the development of effective kinase-targeted therapies.     

Identification of phosphorylation sites within the SH3 domains of Tec family tyrosine kinases

Tec family protein tyrosine kinases (TFKs) play a central role in hematopoietic cellular signaling. Initial activation takes place through specific tyrosine phosphorylation situated in the activation loop. Further activation occurs within the SH3 domain via a transphosphorylation mechanism, which for Bruton's tyrosine kinase (Btk) affects tyrosine 223. We found that TFKs phosphorylate preferentially their own SH3 domains, but differentially phosphorylate other member family SH3 domains, whereas non-related SH3 domains are not phosphorylated. We demonstrate that SH3 domains are good and reliable substrates. We observe that transphosphorylation is selective not only for SH3 domains, but also for dual SH3SH2 domains. However, the dual domain is phosphorylated more effectively. The major phosphorylation sites were identified as conserved tyrosines, for Itk Y180 and for Bmx Y215, both sites being homologous to the Y223 site in Btk. There is, however, one exception because the Tec-SH3 domain is phosphorylated at a non-homologous site, nevertheless a conserved tyrosine, Y206. Consistent with these findings, the 3D structures for SH3 domains point out that these phosphorylated tyrosines are located on the ligand-binding surface. Because a number of Tec family kinases are coexpressed in cells, it is possible that they could regulate the activity of each other through transphosphorylation.     

Activation of Syk protein tyrosine kinase through interaction with integrin beta cytoplasmic domains.

Syk protein tyrosine kinase is essential for immune system development and function [1]and for the maintenance of vascular integrity [2,3]. In leukocytes, Syk is activated by binding to diphosphorylated immune receptor tyrosine-based activation motifs (pITAMs)[1]. Syk can also be activated by integrin adhesion receptors [4,5], but the mechanism of its activation is unknown. Here we report a novel mechanism for Syk's recruitment and activation, which requires that Syk bind to the integrin beta3 cytoplasmic tail. We found that both Syk and the related kinase ZAP-70 bound the beta3 cytoplasmic tail through their tandem SH2 domains. However, unlike Syk binding to pITAMs, this interaction was independent of tyrosine phosphorylation and of the phosphotyrosine binding function of Syk's tandem SH2 domains. Deletion of the four C-terminal residues of the beta3 cytoplasmic tail [beta3(759X)] decreased Syk binding and disrupted its physical association with integrin alphaIIbbeta3. Furthermore, cells expressing alphaIIbbeta3(759X) failed to exhibit Syk activation or lamellipodia formation upon cell adhesion to the alphaIIbbeta3 ligand, fibrinogen. In contrast, FAK phosphorylation and focal adhesion formation were unimpaired by this mutation. Thus, the direct binding of Syk kinase to the integrin beta3 cytoplasmic tail is a novel and functionally significant mechanism for the regulation of this important non-receptor tyrosine kinase.     

Receptor tyrosine kinases.

A major process through which environmental information is transmitted into cells is via activation of protein tyrosine kinases. Receptor tyrosine kinases contain extracellular ligand recognition, single membrane spanning, and cytoplasmic protein tyrosine kinase domains. The cytoplasmic kinase core is flanked by regulatory segments, which in some family members are also inserted into the core kinase domain. Ligand binding initiates receptor signaling from the cell surface. Activated receptors autophosphorylate to remove alternate substrate/inhibitory constraints and to provide loci for assembly of proteins that contain SRC homology regions. Information is transmitted and diffused by tyrosine phosphorylation of the assembled proteins and of cellular substrates that include protein kinases with specificity for serine/threonine residues. Signaling, which is strictly ligand-dependent, is attenuated by down-regulation of receptors and by feed-back inhibitory loops that involve receptor phosphorylation by cellular kinases. The tyrosine kinase receptors are essential for normal growth, development, and reparative processes. Mutations that remove normal regulatory constraints on the approximately 290 amino acid kinase core of these large proteins result in constitutive function and cell transformation.     

Immunofluorescent localization of a 39,000-dalton substrate of tyrosine protein kinases to the cytoplasmic surface of the plasma membrane

The intracellular distribution of p39, a 39,000-dalton substrate for a number of tyrosine protein kinases, has been determined by indirect immunofluorescence microscopy. No binding of anti-p39 antibodies to intact cells was observed, indicating that this protein is not accessible to antibody on the cell surface. Following detergent permeabilization of formaldehyde-fixed cells, a reasonably uniform cytoplasmic labeling was observed. This fluorescence was most pronounced in membrane ruffles, especially in the leading lamellae of migrating cells, and in areas of cell-cell contact. Brief permeabilization of cells with detergent prior to formaldehyde fixation resulted in the appearance of a reticular lattice. An identical staining pattern was observed when fluorescently-labeled lectins were used as plasma membrane markers, but not when antibodies to a variety of cytoskeletal proteins were used. Taken together, these results indicate that p39 is, at least in part, located at the cytoplasmic surface of the plasma membrane. Immunolabeling of Rous sarcoma virus- transformed cells with anti-p39 antibodies resulted in fluorescent staining patterns indistinguishable from those observed in untransformed cells. It is conceivable that p39 plays some structural role within a protein network underlying the plasma membrane.     

Structures of Src-family tyrosine kinases

The crystal structures of three Src-family tyrosine kinases have been determined recently. The structure of the catalytic domain of Lck has been determined in the active autophosphorylated state. The structures of larger constructs of c-Src and Hck, containing the SH3, SH2 and catalytic domains, as well as a C-terminal regulatory tail, have been determined in the down-regulated state, phosphorylated in the C-terminal tail. A comparison of these structures leads to an unanticipated mechanism for the regulation of catalytic activity by cooperative interactions between the SH2, SH3 and catalytic domains.     

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.     

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.     

Trafficking of receptor tyrosine kinases to the nucleus

It has been known for at least 20 years that growth factors induce the internalization of cognate receptor tyrosine kinases (RTKs). The internalized receptors are then sorted to lysosomes or recycled to the cell surface. More recently, data have been published to indicate other intracellular destinations for the internalized RTKs. These include the nucleus, mitochondria, and cytoplasm. Also, it is recognized that trafficking to these novel destinations involves new biochemical mechanisms, such as proteolytic processing or interaction with translocons, and that these trafficking events have a function in signal transduction, implicating the receptor itself as a signaling element between the cell surface and the nucleus.     

Regulation of the c-Abl and Bcr-Abl tyrosine kinases

The prototypic non-receptor tyrosine kinase c-Abl is implicated in various cellular processes. Its oncogenic counterpart, the Bcr-Abl fusion protein, causes certain human leukaemias. Recent insights into the structure and regulation of the c-Abl and Bcr-Abl tyrosine kinases have changed the way we look at these enzymes.     

Structure and function of the protein tyrosine kinases

The protein tyrosine kinases (PTKs) are a large and structurally diverse family of enzymes. The conserved catalytic domain held in common by each member of this family is a self-contained 250–300 amino acid unit bearing sixteen highly conserved linear sequence elements, several of which have been shown to be important to the catalytic activity of this domain. The enzymic activity of the PTKs is clearly an evolutionarily successful theme, and at least 10 distinct morphotypes have been described. Many of these resemble cell surface receptors for growth factors, and for a small sub-set of these receptors a ligand has been discovered. The remainder are located intracellularly and presumably sense and respond to appropriate metabolic cues by exerting their physiologically powerful enzymic activity. A detailed examination of the structure/function relationships of the PTKs and their catalytic domains is particularly revealing in trying to establish the roles that these proteins play in signal transduction in eukaryotic cells.     

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.     

Evolution of thesrc-related protein tyrosine kinases

A phylogenetic analysis ofsrc-related protein tyrosine kinases (PTKs) showed that one group of these genes is quite ancient in the animals, its divergence predating the divergence of the diploblast and triploblast phyla. Three other major groupings of genes were found to predate the divergence of protostome and deuterostome phyla. Most knownsrc-related PTKs of mammals were found to belong to five well-differentiated families: srcA, srcB, abl, csk, and tec. One srcA gene (fyn) has an alternatively spliced seventh exon which shows a different pattern of relationship from the remainder of the gene; this suggests that this exon may have been derived by a recombinational event with another gene, perhaps one related tofgr. The recently published claim that mammalian members of this family expressed in the nervous system evolve more slowly at nonsynonymous nucleotide sites than do those expressed in the immune system was not supported by an analysis of 13 pairs of human and mouse orthologues. Rather, T-cell-specificsrc-related PTKs were found to have higher rates of nonsynonymous substitution than were those having broader expression. This effect was particularly marked in the peptide binding site of the SH2 domain. While the SH2 binding site was highly conserved among paralogous mammalian members of the srcA and srcB subfamilies, no such effect was seen in the comparison of paralogous members of the csk and tec subfamilies. This suggests that, while the peptide binding function of SH2 is conserved within both srcA and srcB subfamilies, paralogous members of the csk and tec subfamilies have diverged functionally with respect to peptide recognition by SH2.