Search PubMed⌕ Search

Biomedical subjects

B J Mayer

Publications and source records attributed to B J Mayer.

At least 37 records · Page 2Linked to original sources
PubMed1998

Dominant-negative mutants of the SH2/SH3 adapters Nck and Grb2 inhibit MAP kinase activation and mesoderm-specific gene induction by eFGF in Xenopus.

The SH2/SH3 adapters Nck, Grb2 and Crk promote the assembly of signaling complexes by binding to tyrosine phosphorylated proteins using their SH2 domains and to proline-rich sequences on effector molecules using their SH3 domains. FGF, which activates a receptor tyrosine kinase, induces mesoderm formation in Xenopus embryos through activation of the Ras/Raf/MAPK signaling pathway. We present evidence that dominant-negative mutants of Nck and Grb2, but not Crk1, can inhibit mesoderm-specific gene induction by eFGF in Xenopus animal cap explants. We also show that dominant-negative mutants of Grb2 and Nck can inhibit eFGF-induced Erk1 activation in Xenopus animal caps, and that targeting the first two SH3 domains of Nck to the membrane can activate Erk1 in the absence of eFGF. Furthermore, combinations of the dominant-negative Grb2 mutants with the inhibitory Nck mutant synergistically inhibited Erk1 activation by eFGF in Xenopus animal caps, suggesting that the dominant-negative Nck and Grb2 mutants inhibit Erk1 activation by binding to different proteins. By contrast only Grb2 mutants could inhibit eFGF-induced Erk1 activation in human 293 cells, demonstrating diversity in the specific mechanisms of signaling from FGF to MAP kinases in different cells.

Adaptor Proteins, Signal Transducing↗
PubMed1998

SH3-Domain binding function of HIV-1 Nef is required for association with a PAK-related kinase.

HIV-1 Nef has previously been shown to bind to Src homology-3 (SH3) domains of a subset of Src family tyrosine kinases. In addition, Nef has been reported to coprecipitate with a serine/threonine kinase activity termed NAK (for Nef-associated kinase). The identity of NAK remains uncertain, but it has been suggested to represent a novel member of the p21-activated kinase (PAK) family. We report here that NAK autophosphorylation is increased not only by an activated form of the p21-family GTPase cdc42 but also by a plasma membrane-targeted fragment of the adapter protein Nck, thus providing further evidence that NAK is related to PAKs. A detailed structure-based mutational analysis of Nef revealed that all amino acid changes that inhibited the Nef/Hck-SH3 interaction, as measured by surface-plasmon resonance, also abolished coprecipitation of NAK. As PAK family proteins do not contain SH3 domains, these observations are best explained by a protein complex in which Nef, NAK, and an SH3-protein all contact each other. In addition, a number of conserved amino acids in Nef that are not involved in SH3 binding were also found to be crucial for association with NAK. Molecular modeling suggests that these residues are involved in formation of an adjacent binding surface for NAK or another critical component of the NAK/Nef complex.

Cell Line↗
PubMed1998

The p35/Cdk5 kinase is a neuron-specific Rac effector that inhibits Pak1 activity.

Cyclin-dependent kinase 5 (Cdk5) and its neuron-specific regulator p35 are essential for neuronal migration and for the laminar configuration of the cerebral cortex. In addition, p35/Cdk5 kinase concentrates at the leading edges of axonal growth cones and regulates neurite outgrowth in cortical neurons in culture. The Rho family of small GTPases is implicated in a range of cellular functions, including cell migration and neurite outgrowth. Here we show that the p35/Cdk5 kinase co-localizes with Rac in neuronal growth cones. Furthermore, p35 associates directly with Rac in a GTP-dependent manner. Another Rac effector, Pak1 kinase, is also present in the Rac-p35/Cdk5 complexes and co-localizes with p35/Cdk5 and Rac at neuronal peripheries. The active p35/Cdk5 kinase causes Pak1 hyperphosphorylation in a Rac-dependent manner, which results in down-regulation of Pak1 kinase activity. Because the Rho family of GTPases and the Pak kinases are implicated in actin polymerization, the modification of Pak1, imposed by the p35/Cdk5 kinase, is likely to have an impact on the dynamics of the reorganization of the actin cytoskeleton in neurons, thus promoting neuronal migration and neurite outgrowth.

Animals↗
PubMed1998

The Wiskott-Aldrich syndrome protein-interacting protein (WIP) binds to the adaptor protein Nck.

Nck is a ubiquitous adaptor molecule composed of three Src homology 3 (SH3) domains followed by a single SH2 domain. Nck links, via its SH2 domain, tyrosine-phosphorylated receptors to effector proteins that contain SH3-binding proline-rich sequences. In this report, we demonstrate that recombinant Nck precipitates endogenous WIP, a novel proline-rich protein that interacts with the Wiskott-Aldrich syndrome protein (WASP), from BJAB cell lysates. Nck binds through its second SH3 domain to WIP, and Nck binds to WIP at a site (amino acids 321-415) that differs from the WASP-binding site (amino acids 416-488). WIP has been shown to associate with the actin polymerization regulatory protein profilin and to induce actin polymerization and cytoskeletal reorganization in lymphoid cells. We demonstrate the presence of profilin in Nck precipitates suggesting that Nck may couple extracellular signals to the cytoskeleton via its interaction with WIP and profilin.

Actins↗
PubMed1997

Signal transduction: clamping down on Src activity.

Recent high-resolution structures of members of the Src family of protein-tyrosine kinases illustrate how a series of cooperative intramolecular interactions represses the catalytic activity of these kinases, but allows for their rapid activation by a variety of regulatory inputs.

Animals↗
PubMed1997

Expression of mutated Nck SH2/SH3 adaptor respecifies mesodermal cell fate in Xenopus laevis development.

Nck is a widely expressed SH2/SH3 adaptor protein containing one SH2 and three SH3 domains. Although Nck is assumed to mediate the formation of protein-protein complexes during signaling, little is currently known about its specific function. We have constructed a series of Nck SH3 and SH2 domain mutants, expressed them in Xenopus laevis embryos, and monitored injected embryos for developmental abnormalities. This approach allows correlation of developmental phenotypes with the presence or absence of specific Nck protein-binding domains. We show that microinjection of RNA-encoding Nck with an inactivating mutation in the third SH3 domain (NckK229) into dorsal blastomeres of early embryos caused anterior truncation with high frequency, and membrane localization of both the first and second SH3 domains together was sufficient to induce this anterior-truncation phenotype. Molecular marker analysis of explants revealed that the expression of NckK229 ventralized dorsal mesoderm. Lineage tracing experiments demonstrated that the expression of Nck K229 in dorsal blastomeres affected the migratory properties of mesoderm cells in gastrulation and led to the adoption of a more posterior fate. These data suggest that protein(s) that bind the first and second SH3 domains of Nck can affect the response to signals that establish dorso-ventral patterning, and that protein(s) that bind the third SH3 domain antagonize the ventralizing effect of the first two SH3 domains.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Activation of Pak by membrane localization mediated by an SH3 domain from the adaptor protein Nck.

BACKGROUND: The adaptor protein Nck consists of three Src homology 3 (SH3) domains followed by one SH2 domain. Like the Grb2 adaptor protein, which is known to couple receptor tyrosine kinases to the small GTPase Ras, Nck is presumed to bind to tyrosine-phosphorylated proteins using its SH2 domain and to downstream effector proteins using its SH3 domain. Little is known, however, about the specific biological function of Nck. The Pak family of serine/threonine kinases are known to be activated by binding to the GTP-bound form of Cdc42 or Rac1, which are small GTPases of the Rho family that are involved in regulating the organization of the actin cytoskeleton. RESULTS: We present evidence that Nck can mediate the relocalization and subsequent activation of the Pak1 kinases. We show that Nck associates in vivo with Pak using the second of its three SH3 domains, and that localization of this individual Nck SH3 domain, or of Pak kinase itself, to the membrane results in activation of Pak and stimulation of downstream mitogen activated protein kinase cascades. Activation of downstream signaling by the membrane-localized Nck SH3 domain is blocked by a kinase-inactive mutant form of Pak1. CONCLUSION: These results demonstrate that localization of Pak1 to the membrane in the absence of other signals is sufficient for its activation, and imply that the Nck adaptor protein could function to link changes in tyrosine phosphorylation of cellular proteins to the Cdc42/Pak signaling pathway.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Crk is required for apoptosis in Xenopus egg extracts.

Apoptosis is essential for the development and homeostasis of multicellular organisms. Recently, a cell-free extract prepared from Xenopus eggs was shown to recapitulate intracellular apoptotic pathways in vitro. While many stimuli have been shown to trigger apoptosis in a variety of cell types, the intracellular signaling pathways involved in apoptosis remain largely unknown. Here we show that addition of a recombinant protein containing the phosphotyrosine binding (SH2) domain from the adaptor protein crk, but not those derived from a panel of other signaling proteins, can prevent apoptosis in the Xenopus egg extract system. Furthermore, immunodepletion of endogenous crk protein from the egg extracts, or addition of anti-crk antisera to these extracts, prevents apoptosis. The ability to undergo apoptosis can be restored to these extracts by addition of recombinant crk protein. These results directly demonstrate that crk participates in apoptotic signaling.

Amino Acid Sequence↗
PubMed1997

p21CIP1 and Cdc25A: competition between an inhibitor and an activator of cyclin-dependent kinases.

Cdc25A, a phosphatase essential for G1-S transition, associates with, dephosphorylates, and activates the cell cycle kinase cyclin E-cdk2. p21CIP1 and p27 are cyclin-dependent kinase (cdk) inhibitors induced by growth-suppressive signals such as p53 and transforming growth factor beta (TGF-beta). We have identified a cyclin binding motif near the N terminus of Cdc25A that is similar to the cyclin binding Cy (or RR LFG) motif of the p21CIP1 family of cdk inhibitors and separate from the catalytic domain. Mutations in this motif disrupt the association of Cdc25A with cyclin E- or cyclin A-cdk2 in vitro and in vivo and selectively interfere with the dephosphorylation of cyclin E-cdk2. A peptide based on the Cy motif of p21 competitively disrupts the association of Cdc25A with cyclin-cdks and inhibits the dephosphorylation of the kinase. p21 inhibits Cdc25A-cyclin-cdk2 association and the dephosphorylation of cdk2. Conversely, Cdc25A, which is itself an oncogene up-regulated by the Myc oncogene, associates with cyclin-cdk and protects it from inhibition by p21. Cdc25A also protects DNA replication in Xenopus egg extracts from inhibition by p21. These results describe a mechanism by which the Myc- or Cdc25A-induced oncogenic and p53- or TGF-beta-induced growth-suppressive pathways counterbalance each other by competing for cyclin-cdks.

Animals↗
PubMed1996

Growth inhibition by Abl requires an interplay of its SH2 and tyrosine kinase domains.

Overexpression of c-Abl tyrosine kinase can be growth inhibitory in certain fibroblast cell lines. Using a series of conditional chimeras between Abl and Src, we have now further dissected the Abl protein to determine which domains are required for this function. We found that growth inhibition, unlike transformation by oncogenic forms of Abl, is dependent on the presence of the cognate SH2 and tyrosine kinase domains. Since growth inhibition correlates with low tyrosine kinase activity, it may involve highly specific interactions of target proteins with both domains without the processivity of phosphorylation associated with oncogenic Abl.

3T3 Cells↗
PubMed1995

Why two heads are better.

The tandem SH2 domains of the ZAP-70 kinase complexed with a doubly phosphorylated ligand reveal a mechanism for building a high-affinity interaction with very low non-specific binding.

CD3 Complex↗
PubMed1995

The solution structure of Abl SH3, and its relationship to SH2 in the SH(32) construct.

BACKGROUND: The Src homology domains, SH3 and SH2, of Abl protein tyrosine kinase regulate enzymatic activity in vivo. Abl SH3 suppresses kinase activity, whereas Abl SH2 is required for the transforming activity of the activated form of Abl. We expect that the solution structures of Abl SH3, Abl SH2 and Abl SH(32) (a dual domain comprising SH3 and SH2 subdomains) will contribute to a structural basis for understanding the mechanism of the Abl 'regulatory apparatus'. RESULTS: We present the solution structure of the free Abl SH3 domain and a structural characterization of the Abl regulatory apparatus, the SH(32) dual domain. The solution structure of Abl SH3 was determined using multidimensional double resonance NMR spectroscopy. It consists of two antiparallel beta sheets packed orthogonally, an arrangement first shown in spectrin SH3. Compared with the crystal structure of the Abl SH3 complexed with a natural ligand, there is no significant difference in overall folding pattern. The structure of the Abl SH(32) dual domain was characterized by NMR spectroscopy using the 1H and 15N resonance assignment of Abl SH3 and Abl SH2. On the basis of the high degree of similarity in chemical shifts and hydrogen/deuterium exchange pattern for the individual domains of SH3 and SH2 compared with those of the SH(32) dual domain, a structural model of the Abl SH(32) regulatory apparatus is suggested. This model is in good agreement with the ligand-binding characteristics of Abl SH3, SH2 and SH(32). The binding constants for isolated SH3 and SH2 domains when binding to natural ligands, measured by intrinsic fluorescence quenching, do not differ significantly from the constants of these domains within SH(32). CONCLUSION: The solution structures of free Abl SH3 and Abl SH2, and the structural model of Abl SH(32), provide information about the overall topology of these modular domains. The structural model of Abl SH(32), a monomer, consists of the SH3 and SH2 domains connected by a flexible linker. Sites of ligand binding for the two subdomains are independent.

Amino Acid Sequence↗
PubMed1995

SH3 domains. Minding your p's and q's.

SH3 domains mediate many important protein-protein interactions. The molecular basis of the binding of these domains to their ligands has been revealed, making it possible to identify SH3-binding sites in new proteins.

Amino Acid Sequence↗
PubMed1995

Evidence that SH2 domains promote processive phosphorylation by protein-tyrosine kinases.

BACKGROUND: Non-receptor protein-tyrosine kinases often contain at least one Src homology 2 (SH2) domain, a protein module that binds with high affinity to tyrosine-phosphorylated peptides. Because SH2 domains would be predicted to bind with high affinity to proteins phosphorylated by the kinase, but not to the unphosphorylated substrate, their presence in tyrosine kinases has been puzzling. An important role for the SH2 domain of the Abl tyrosine kinase was suggested by work showing that Abl requires an intact SH2 domain in order to malignantly transform cells, and that replacement of the Abl SH2 domain with heterologous SH2 domains alters the spectrum of proteins phosphorylated detectably by Abl in vivo. RESULTS: We have used purified wild-type and mutant Abl kinases to examine the roles of the Abl's SH2 and catalytic domains in phosphorylation of p130CAS, a model substrate that has multiple potential phosphorylation sites. We find that an SH2 domain is required for efficient hyperphosphorylation of p130 in vitro. We use chimeric mutants with heterologous SH2 domains to demonstrate that the SH2 domain of the oncogenically transforming adaptor protein Crk, which is the SH2 domain predicted to bind with highest affinity (of those tested) to potential phosphorylation sites in p130, is best able to facilitate hyperphosphorylation. This is the case whether the catalytic domain of the kinase is derived from Abl or from its distant relative, Src. These studies also reveal a role for binding of Crk to Abl in mediating phosphorylation by the kinase. Using purified proteins, we demonstrate that association with Crk strikingly enhances the ability of Abl to hyperphosphorylate p130. There is an excellent correlation between the ability of mutant Crk proteins to promote hyperphosphorylation of p130 by Abl and their ability to transform rodent fibroblasts. CONCLUSION: Our data suggest that, ultimately, the substrate specificity of a non-receptor tyrosine kinase is dependent on the binding specificity of its associated SH2 domain. The SH2 domain binds tightly to a subset of proteins phosphorylated by the catalytic domain, leading to processive phosphorylation of those proteins. Substrate specificity can be broadened by an association between the kinase and proteins, such as Crk, that contain additional SH2 domains; this may play a role in malignant transformation by Crk.

Animals↗
PubMed1995

Differential inhibition of signaling pathways by dominant-negative SH2/SH3 adapter proteins.

SH2/SH3 adapters are thought to function in signal transduction pathways by coupling inputs from tyrosine kinases to downstream effectors such as Ras. Members of the mitogen-activated protein kinase family are known to be activated by a variety of mitogenic stimuli, including tyrosine kinases such as Abl and the epidermal growth factor (EGF) receptor. We have used activation of the mitogen-activated protein kinase Erk-1 as a model system with which to examine whether various dominant-negative SH2/SH3 adapters (Grb2, Crk, and Nck) could block signaling pathways leading to Erk activation. Activation of Erk-1 by oncogenic Abl was effectively inhibited by Grb2 with mutations in either its SH2 or SH3 domain or by Crk-1 with an SH3 domain mutation. The Crk-1 SH2 mutant was less effective, while Nck SH2 and SH3 mutants had little or no effect on Erk activation. These results suggest that both Crk and Grb2 may contribute to the activation of Erk by oncogenic Abl, whereas Nck is unlikely to participate in this pathway. Next we examined whether combinations of these dominant-negative adapters could inhibit Erk activation more effectively than each mutant alone. When combinations of Crk-1 and Grb2 mutants were analyzed, the combination of the Crk-1 SH3 mutant plus the Grb2 SH3 mutant gave a striking synergistic effect. This finding suggests that in Abl-transformed cells, more than one class of tyrosine-phosphorylated sites (those that bind the Grb2 SH2 domain and those that bind the Crk SH2 domain) can lead to Ras activation. In contrast to results with Abl, Erk activation by EGF was strongly inhibited only by Grb2 mutants; Crk and Nck mutants had little or no effect. This finding suggests that Grb2 is the only adapter involved in the activation of Erk by EGF. Dominant-negative adaptors provide a novel means to identify binding interactions important in vivo for signaling in response to a variety of stimuli.

Adaptor Proteins, Signal Transducing↗
PubMed1994

Mutagenic analysis of the roles of SH2 and SH3 domains in regulation of the Abl tyrosine kinase.

We have used in vitro mutagenesis to examine in detail the roles of two modular protein domains, SH2 and SH3, in the regulation of the Abl tyrosine kinase. As previously shown, the SH3 domain suppresses an intrinsic transforming activity of the normally nontransforming c-Abl product in vivo. We show here that this inhibitory activity is extremely position sensitive, because mutants in which the position of the SH3 domain within the protein is subtly altered are fully transforming. In contrast to the case in vivo, the SH3 domain has no effect on the in vitro kinase activity of the purified protein. These results are consistent with a model in which the SH3 domain binds a cellular inhibitory factor, which in turn must physically interact with other parts of the kinase. Unlike the SH3 domain, the SH2 domain is required for transforming activity of activated Abl alleles. We demonstrate that SH2 domains from other proteins (Ras-GTPase-activating protein, Src, p85 phosphatidylinositol 3-kinase subunit, and Crk) can complement the absence of the Abl SH2 domain and that mutants with heterologous SH2 domains induce altered patterns of tyrosine-phosphorylated proteins in vivo. The positive function of the SH2 domain is relatively position independent, and the effect of multiple SH2 domains appears to be additive. These results suggest a novel mechanism for regulation of tyrosine kinases in which the SH2 domain binds to, and thereby enhances the phosphorylation of, a subset of proteins phosphorylated by the catalytic domain. Our data also suggest that the roles of the SH2 and SH3 domains in the regulation of Abl are different in several respects from the roles proposed for these domains in the closely related Src family of tyrosine kinases.

Animals↗