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.     

The linker between SH2 and kinase domains positively regulates catalysis of the Tec family kinases

Tec family nonreceptor tyrosine kinases are key immunological enzymes that control processes that range from T and B cell development to reorganization of the actin cytoskeleton. The full-length Tec kinases have been resistant to crystallization. This lack of structural data and the paucity of in vitro biochemical data for this kinase family leave a void in our understanding of Tec kinase regulation. In this report we have used interleukin-2 tyrosine kinase (Itk) as a model system to gain insight into the regulatory apparatus of the Tec kinases. Use of a quantitative in vitro kinase assay has uncovered an essential role for the short linker region flanked by the SH2 and kinase domains of Itk in positively regulating Itk catalytic activity. The precise residues that allosterically regulate Itk are conserved among Tec kinases, pointing to the conserved nature of this regulatory mechanism within the family. These findings indicate that Tec kinases are not regulated in the same manner as the Src kinases but rather share some of the regulatory features of Csk instead.     

Tec family kinases

Tec family kinases (TFKs) are ancient kinases, vital for the development of both mammals and fruit flies. The first TFKs were described 20 years ago, and one of them, BTK, was found to be vital for B-lymphocyte development. However, during the last five years several seminal discoveries have been made, significantly advancing our understanding of these signal transducers.     

Dynamics of the Tec‐family tyrosine kinase SH3 domains

The Src Homology 3 (SH3) domain is an important regulatory domain found in many signaling proteins. X‐ray crystallography and NMR structures of SH3 domains are generally conserved but other studies indicate that protein flexibility and dynamics are not. We previously reported that based on hydrogen exchange mass spectrometry (HX MS) studies, there is variable flexibility and dynamics among the SH3 domains of the Src‐family tyrosine kinases and related proteins. Here we have extended our studies to the SH3 domains of the Tec family tyrosine kinases (Itk, Btk, Tec, Txk, Bmx). The SH3 domains of members of this family augment the variety in dynamics observed in previous SH3 domains. Txk and Bmx SH3 were found to be highly dynamic in solution by HX MS and Bmx was unstructured by NMR. Itk and Btk SH3 underwent a clear EX1 cooperative unfolding event, which was localized using pepsin digestion and mass spectrometry after hydrogen exchange labeling. The unfolding was localized to peptide regions that had been previously identified in the Src‐family and related protein SH3 domains, yet the kinetics of unfolding were not. Sequence alignment does not provide an easy explanation for the observed dynamics behavior, yet the similarity of location of EX1 unfolding suggests that higher‐order structural properties may play a role. While the exact reason for such dynamics is not clear, such motions can be exploited in intra‐ and intermolecular binding assays of proteins containing the domains.     

SH2-B family members differentially regulate JAK family tyrosine kinases.

Activation of JAK tyrosine kinases is an essential step in cell signaling by multiple hormones, cytokines, and growth factors, including growth hormone (GH) and interferon-gamma. Previously, we identified SH2-B beta as a potent activator of JAK2 (Rui, L., and Carter-Su, C. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 7172-7177). Here, we investigated whether the activation of JAK2 by SH2-B beta is specific to JAK2 and SH2-B beta or extends to other JAKs or other members of the SH2-B beta family. When SH2-B beta was overexpressed with JAK1 or JAK3, SH2-B beta failed to increase their activity. However, SH2-B beta bound to both and was tyrosyl-phosphorylated by JAK1. In contrast to SH2-B beta, APS decreased tyrosyl phosphorylation of GH-stimulated JAK2 as well as Stat5B, a substrate of JAK2. APS also decreased tyrosyl phosphorylation of JAK1, but did not affect the activity or tyrosyl phosphorylation of JAK3. Overexpressed APS bound to and was tyrosyl-phosphorylated by all three JAKs. Consistent with these data, in 3T3-F442A adipocytes, endogenous APS was tyrosyl-phosphorylated in response to GH and interferon-gamma. These results suggest that 1) SH2-B beta specifically activates JAK2, 2) APS negatively regulates both JAK2 and JAK1, and 3) both SH2-B beta and APS may serve as adapter proteins for all three JAKs independent of any role they have in JAK activity.     

Dynamic coupling between the SH2 and SH3 domains of c-Src and Hck underlies their inactivation by C-terminal tyrosine phosphorylation

The effect of C-terminal tyrosine phosphorylation on molecular motions in the Src kinases Hck and c-Src is investigated by molecular dynamics simulations. The SH2 and SH3 domains of the inactive kinases are seen to be tightly coupled by the connector between them, impeding activation. Dephosphorylation of the tail reduces the coupling between the SH2 and SH3 domains in the simulations, as does replacement of connector residues with glycine. A mutational analysis of c-Src expressed in Schizosaccharomyces pombe demonstrates that replacement of residues in the SH2-SH3 connector with glycine activates c-Src. The SH2-SH3 connector appears to be an inducible "snap lock" that clamps the SH2 and SH3 domains upon tail phosphorylation, but which allows flexibility when the tail is released.     

Identification of Mcm2 phosphorylation sites by S-phase-regulating kinases

Minichromosome maintenance 2-7 proteins play a pivotal role in replication of the genome in eukaryotic organisms. Upon entry into S-phase several subunits of the MCM hexameric complex are phosphorylated. It is thought that phosphorylation activates the intrinsic MCM DNA helicase activity, thus allowing formation of active replication forks. Cdc7, Cdk2, and ataxia telangiectasia and Rad3-related kinases regulate S-phase entry and S-phase progression and are known to phosphorylate the Mcm2 subunit. In this work, by in vitro kinase reactions and mass spectrometry analysis of the products, we have mapped phosphorylation sites in the N terminus of Mcm2 by Cdc7, Cdk2, Cdk1, and CK2. We found that Cdc7 phosphorylates Mcm2 in at least three different sites, one of which corresponds to a site also reported to be phosphorylated by ataxia telangiectasia and Rad3-related. Three serine/proline sites were identified for Cdk2 and Cdk1, and a unique site was phosphorylated by CK2. We raised specific anti-phosphopeptide antibodies and found that all the sites identified in vitro are also phosphorylated in cells. Importantly, although all the Cdc7-dependent Mcm2 phosphosites fluctuate during the cell cycle with kinetics similar to Cdc7 kinase activity and Cdc7 protein levels, phosphorylation of Mcm2 in the putative cyclin-dependent kinase (Cdk) consensus sites is constant during the cell cycle. Furthermore, our analysis indicates that the majority of the Mcm2 isoforms phosphorylated by Cdc7 are not stably associated with chromatin. This study forms the basis for understanding how MCM functions are regulated by multiple kinases within the cell cycle and in response to external perturbations.     

Crystal structure of the SH3 domain in human Fyn; comparison of the three-dimensional structures of SH3 domains in tyrosine kinases and spectrin.

The Src-homology 3 (SH3) region is a protein domain consisting of approximately 60 residues. It occurs in a large number of eukaryotic proteins involved in signal transduction, cell polarization and membrane--cytoskeleton interactions. The function is unknown, but it is probably involved in specific protein--protein interactions. Here we report the crystal structure of the SH3 domain of Fyn (a Src family tyrosine kinase) at 1.9 A resolution. The crystals have two SH3 molecules per asymmetric unit. These two Fyn SH3 domains are not related by a local twofold axis. The crystal structures of spectrin and Fyn SH3 domains as well as the solution structure of the Src SH3 domain show that these all have the same basic fold. A protein domain which has the same topology as SH3 is present in the prokaryotic regulatory enzyme BirA. The comparison between the crystal structures of Fyn and spectrin SH3 domains shows that a conserved surface patch, consisting mainly of aromatic residues, is flanked by two hairpin-like loops (residues 94-104 and 114-118 in Fyn). These loops are different in tyrosine kinase and spectrin SH3 domains. They could modulate the binding properties of the aromatic surface.     

Peroxynitrite activates kinases of the src family and upregulates tyrosine phosphorylation signaling

The hypothesis that peroxynitrite may act as a signaling molecule able to upregulate protein tyrosine phosphorylation is discussed. This article focuses on the mechanisms for activating kinases of the src family, an important class of nonreceptor tyrosine kinases implicated in the regulation of cell communication, proliferation, migration, differentiation, and survival. Recent in vitro findings show that in erythrocytes, synaptosomes, and cerebellar primary culture cells peroxynitrite is able to inhibit phosphatases and to activate different members of the src kinase family through different mechanisms involving cysteine-dependent and -independent processes. The ability of nitrotyrosine-containing peptides with SH2 binding affinity to activate src kinases is also discussed.     

Tec kinases mediate sustained calcium influx via site-specific tyrosine phosphorylation of the phospholipase Cgamma Src homology 2-Src homology 3 linker

Tyrosine phosphorylation of phospholipase Cgamma2 (PLCgamma2) is a crucial activation switch that initiates and maintains intracellular calcium mobilization in response to B cell antigen receptor (BCR) engagement. Although members from three distinct families of non-receptor tyrosine kinases can phosphorylate PLCgamma in vitro, the specific kinase(s) controlling BCR-dependent PLCgamma activation in vivo remains unknown. Bruton's tyrosine kinase (Btk)-deficient human B cells exhibit diminished inositol 1,4,5-trisphosphate production and calcium signaling despite a normal inducible level of total PLCgamma2 tyrosine phosphorylation. This suggested that Btk might modify a critical subset of residues essential for PLCgamma2 activity. To evaluate this hypothesis, we generated site-specific phosphotyrosine antibodies recognizing four putative regulatory residues within PLCgamma2. Whereas all four sites were rapidly modified in response to BCR engagement in normal B cells, Btk-deficient B cells exhibited a marked reduction in phosphorylation of the Src homology 2 (SH2)-SH3 linker region sites, Tyr(753) and Tyr(759). Phosphorylation of both sites was restored by expression of Tec, but not Syk, family kinases. In contrast, phosphorylation of the PLCgamma2 carboxyl-terminal sites, Tyr(1197) and Tyr(1217), was unaffected by the absence of functional Btk. Together, these data support a model whereby Btk/Tec kinases control sustained calcium signaling via site-specific phosphorylation of key residues within the PLCgamma2 SH2-SH3 linker.     

Identification of the insulin-responsive tyrosine phosphorylation sites on IRSp53

The 53-kDa insulin receptor substrate protein (IRSp53) is part of a regulatory network that organises the actin cytoskeleton in response to stimulation by small GTPases, promoting formation of actin-rich cell protrusions such as filopodia and lamellipodia. It had been established earlier that IRSp53 is tyrosine phosphorylated in response to stimulation of the insulin and insulin-related growth factor receptors, but the consequences of tyrosine phosphorylation for IRSp53 function are unknown. Here, we have used a variety of IRSp53 truncation and point mutants to identify insulin-responsive tyrosine phosphorylation sites on IRSp53. We have found that the C-terminal half of IRSp53 (residues 251-521) undergoes tyrosine phosphorylation in response to insulin stimulation of the insulin beta receptor or epidermal growth factor stimulation via the epidermal growth factor receptor, and that the key residue for insulin receptor-mediated phosphorylation is tyrosine 310, located in a region between the N-terminal IRSp53/MIM homology domain (IMD, residue 1-250) and the central SH3 domain (residues 374-438) that is predicted to be natively unstructured. Mutation of tyrosine 310 to phenylalanine or glutamic acid abrogates the phosphorylation in response to insulin stimulation, but not in response to stimulation of the epidermal growth factor receptor. The N-terminal IMD, which mediates dimerisation of IRSp53, is required for efficient tyrosine phosphorylation downstream of either the insulin or epidermal growth factor receptor stimulation, yet does not appear to include a tyrosine-phosphorylated site itself. Thus, we have identified tyrosine 310 as a primary site of tyrosine phosphorylation in response to insulin signalling and we have shown that although IRSp53 is tyrosine phosphorylated in response to epidermal growth factor receptor signalling, tyrosine 310 is not crucial. Furthermore, the tyrosine phosphorylation status does not appear to affect the cell morphology and production of filopod-like structures upon expression of IRSp53.     

The Tec family of tyrosine kinases in T cells,amplifiers of T cell receptor signals

ITK and Rlk/Txk are the predominant Tec family of tyrosine kinases expressed in T cells, and are involved in T cell antigen receptor mediated activation of T cells. These kinases require prior activation of Lck, Zap-70 and PI3-kinase for efficient activation. They share major substrates with both Lck and Zap-70, however the pathways they regulate are unclear. Recent evidence suggests that these kinases may not activate unique pathways, but instead serve as amplifiers for the upstream kinases Lck and Zap-70. This review will discuss the evidence for this view.     

SH2-Dependent Autophosphorylation within the Tec Family Kinase Itk

The Tec family kinase, Itk (interleukin-2 tyrosine kinase), undergoes an in cis autophosphorylation on Y180 within its Src homology 3 (SH3) domain. Autophosphorylation of the Itk SH3 domain by the Itk kinase domain is strictly dependent on the presence of the intervening Src homology 2 (SH2) domain. A direct docking interaction between the Itk kinase and SH2 domains brings the Itk SH3 domain into the active site where Y180 is then phosphorylated. We now identify the residues on the surface of the Itk SH2 domain responsible for substrate docking and show that this SH2 surface mediates autophosphorylation in the full-length Itk molecule. The canonical phospholigand binding site on the SH2 domain is not involved in substrate docking, instead the docking site consists of side chains from three loop regions (AB, EF and BG) and part of the βD strand. These results are extended into Btk (Bruton's tyrosine kinase), a Tec family kinase linked to the B-cell deficiency X-linked agammaglobulinemia (XLA). Our results suggest that some XLA-causing mutations might impair Btk phosphorylation.     

Physical and functional interactions between SH2 and SH3 domains of the Src family protein tyrosine kinase p59fyn.

The Src family protein tyrosine kinases participate in signalling through cell surface receptors that lack intrinsic tyrosine kinase domains. All nine members of this family possess adjacent Src homology (SH2 and SH3) domains, both of which are essential for repression of the enzymatic activity. The repression is mediated by binding between the SH2 domain and a C-terminal phosphotyrosine, and the SH3 domain is required for this interaction. However, the biochemical basis of functional SH2-SH3 interaction is unclear. Here, we demonstrate that when the SH2 and SH3 domains of p59fyn (Fyn) were present as adjacent domains in a single protein, binding of phosphotyrosyl peptides and proteins to the SH2 domain was enhanced, whereas binding of a subset of cellular polypeptide ligands to the SH3 domain was decreased. An interdomain communication was further revealed by occupancy with domain-specific peptide ligands: occupancy of the SH3 domain with a proline-rich peptide enhanced phosphotyrosine binding to the linked SH2 domain, and occupancy of the SH2 domain with phosphotyrosyl peptides enhanced binding of certain SH3-specific cellular polypeptides. Second, we demonstrate a direct binding between purified SH2 and SH3 domains of Fyn and Lck Src family kinases. Heterologous binding between SH2 and SH3 domains of closely related members of the Src family, namely, Fyn, Lck, and Src, was also observed. In contrast, Grb2, Crk, Abl, p85 phosphatidylinositol 3-kinase, and GTPase-activating protein SH2 domains showed lower or no binding to Fyn or Lck SH3 domains. SH2-SH3 binding did not require an intact phosphotyrosine binding pocket on the SH2 domain; however, perturbations of the SH2 domain induced by specific high-affinity phosphotyrosyl peptide binding abrogated binding of the SH3 domain. SH3-SH2 binding was observed in the presence of proline-rich peptides or when a point mutation (W119K) was introduced in the putative ligand-binding pouch of the Fyn SH3 domain, although these treatments completely abolished the binding to p85 phosphatidylinositol 3-kinase and other SH3-specific polypeptides. These biochemical SH2-SH3 interactions suggest novel mechanisms of regulating the enzymatic activity of Src kinases and their interactions with other proteins.     

Regulation of actin dynamics by tyrosine phosphorylation: identification of tyrosine phosphorylation sites within the actin-severing domain of villin

We have previously shown that villin, an epithelial cell actin-binding protein, is tyrosine phosphorylated both in vitro and in vivo and that villin's actin-modifying functions are regulated by phosphorylation. Here as a first step toward understanding the role of villin tyrosine phosphorylation, we sought to identify the major phosphorylation site(s) in human villin and study its role in actin filament assembly. We generated a series of carboxyl-terminal truncation mutants of villin and cloned them in the prokaryotic expression vector pGEX-2T. Full-length villin and the truncation mutants were expressed in TKX1 cells, which carry an inducible tyrosine kinase gene. Using this approach, we identified a region in the amino-terminal actin-severing domain of villin as the site of phosphorylation (amino acids 1-261). Five phosphorylation sites were identified by direct mutation of candidate tyrosines (Y) to phenylalanine (F), namely, Y46, -60, -64, -81, and -256. Changing all of these sites to phenylalanine resulted in a villin mutant that neither was phosphorylated in TKX1 cells nor was a substrate for c-src kinase in an in vitro kinase assay. Using a pyrene actin-based fluorescence assay, we mapped the various phosphorylated tyrosine residues with the actin-nucleating and -depolymerizing functions of villin. Phosphorylation of any one of the identified sites inhibited the actin-nucleating function of villin, whereas phosphorylation at Y46 and/or Y60 increased the actin-severing activity of villin. Since there is significant homology between the amino-terminal end of villin and other actin-severing proteins, the results provide a structural basis for the actin-severing mechanism and help understand the relationship of phosphorylation with this function.     

The SH2 and SH3 adapter Nck: a two-gene family and a linker between tyrosine kinases and multiple signaling networks

SH2 and SH3 adapter proteins connect cell surface tyrosine kinases to intracellular signaling networks. For instance, the SH3-SH2-SH3 adapter Grb2 links receptor tyrosine kinases to the Ras pathway. Nck, composed of three SH3 domains and one SH2 domain, represents a two-gene (alpha and beta) family in mammals. Nckalpha and Nckbeta are expressed in the same cells and appear to have distinct signaling specificity. Studies show that Nck plays a role in cell mitogenesis and morphogenesis. The former uses Ras-dependent and Ras-independent pathways. The latter appears to coordinate with the Cdc42/Rac>PAK1/WASp>actin cytoskeleton pathway. Understanding the specificity of Nckalpha and Nckbeta signal transduction will provide answers for the previously often conflicting observations.     

The Tec family of cytoplasmic tyrosine kinases: mammalian Btk, Bmx, Itk, Tec, Txk and homologs in other species

Cytoplasmic protein-tyrosine kinases (PTKs) are enzymes involved in transducing a vast number of signals in metazoans. The importance of the Tec family of kinases was immediately recognized when, in 1993, mutations in the gene encoding Bruton's tyrosine kinase (Btk) were reported to cause the human disease X-linked agammaglobulinemia (XLA). Since then, additional kinases belonging to this family have been isolated, and the availability of full genome sequences allows identification of all members in selected species enabling phylogenetic considerations. Tec kinases are endowed with Pleckstrin homology (PH) and Tec homology (TH) domains and are involved in diverse biological processes related to the control of survival and differentiation fate. Membrane translocation resulting in the activation of Tec kinases with subsequent Ca2+ release seems to be a general feature. However, nuclear translocation may also be of importance. The purpose of this essay is to characterize members of the Tec family and discuss their involvement in signaling. The three-dimensional structure, expression pattern and evolutionary aspects will also be considered.     

Inhibition of protein phosphorylation modulates expression of the Jak family protein tyrosine kinases.

Treatment of murine Friend cells with a dose of the protein kinase inhibitor staurosporine, which is able to block the response of the cells to interferons, appears to inhibit phosphorylation of Jak proteins and, interestingly, to specifically reduce tyk2 and Jak1 expression and to increase Jak2 both in the presence and in the absence of interferons. Therefore, a potential role for phosphorylation events in the regulation of expression of the Jak family members is suggested.