Interaction of two proline-rich sequences of cell adhesion kinase β with SH3 domains of p130Cas-related proteins and a GTPase-activating protein, Graf

Abstract Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia are caused by mutations of the WAS protein (WASP) gene. WASP may be involved in the regulation of podosome, an actin-rich dynamic cell adhesion structure formed by various types of cells. The molecular links between WASP and podosomes or other cell adhesion structures are unknown. Platelets express an SH2-SH3 adapter molecule, CrkL, that can directly associate with paxillin, which is localized in podosomes. The hypothesis that CrkL binds to WASP was, therefore, tested. Results from coprecipitation experiments using anti-CrkL and GST-fusion proteins suggest that CrkL binds to WASP through its SH3 domain and that the binding was not affected by WASP tyrosine phosphorylation. The binding of GST-fusion SH3 domain of PSTPIP1 in vitro was also not affected by WASP tyrosine phosphorylation, suggesting that the binding of the SH3 domains to WASP is not inhibited by tyrosine phosphorylation of WASP. Anti-CrkL also coprecipitates a 72-kd protein, which was identified as syk tyrosine kinase, critical for collagen induced-platelet activation. CrkL immunoprecipitates contain kinase-active syk, as evidenced by an in vitro kinase assay. Coprecipitation experiments using GST-fusion CrkL proteins suggest that both SH2 and SH3 domains of CrkL are involved in the binding of CrkL to syk. WASP, CrkL, syk, and paxillin-like Hic-5 incorporated to platelet cytoskeleton after platelet aggregation. Thus, CrkL is a novel molecular adapter for WASP and syk and may potentially transfer these molecules to the cytoskeleton through association with cytoskeletal proteins such as Hic-5.

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

Molecular and Cellular Biology ◽

10.1128/mcb.14.5.2883 ◽

1994 ◽

Vol 14 (5) ◽

pp. 2883-2894 ◽

Cited By ~ 127

Author(s):

B J Mayer ◽

D Baltimore

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Kinases ◽

Sh3 Domain ◽

Sh2 Domain ◽

Sh3 Domains ◽

Sh2 Domains ◽

Abl Tyrosine Kinase ◽

Transforming Activity

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.

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Interaction of Ash/Grb-2 via its SH3 domains with neuron-specific p150 and p65

Biochemical Journal ◽

10.1042/bj3160639 ◽

1996 ◽

Vol 316 (2) ◽

pp. 639-645 ◽

Cited By ~ 10

Author(s):

Kenji MIURA ◽

Hiroaki MIKI ◽

Kuniko SHIMAZAKI ◽

Nobufumi KAWAI ◽

Tadaomi TAKENAWA

Keyword(s):

Growth Factor Receptor ◽

Sh3 Domain ◽

Bovine Brain ◽

Amino Acid Sequences ◽

Binding Motif ◽

Glutathione S Transferase ◽

Western Blots ◽

Sh3 Domains ◽

Epidermal Growth

We found that 180 kDa, 150 kDa (p150), 110 kDa, 100 kDa and 65 kDa (p65) proteins comprise the major Ash/Grb-2-binding proteins in bovine brain. Among these proteins, 180 kDa and 100 kDa proteins have already been identified as Sos and dynamin respectively. Here, p150 and p65 were affinity-purified with glutathione S-transferase–Ash fusion protein and their partial amino acid sequences were determined. Analysis showed p150 and p65 to be new proteins. These two proteins bind to both the N-terminal SH3 domain and the C-terminal SH3 domain of Ash. It was found that p150 and p65 are expressed predominantly in brain, although Ash is widely distributed in all tissues examined by Western blots. Immunohistochemical staining of rat brain showed p150 and p65 to be localized in a variety of neurons in the cerebellum and hippocampus, with p65 being especially concentrated in the nerve terminal. When the Ash-binding-motif peptide of the epidermal growth factor receptor was used to detect complexes formed with Ash in vivo, 180 kDa, 150 kDa, 110 kDa, 100 kDa and 65 kDa proteins were also bound; this shows that these proteins form complexes with Ash in brain. In addition, p150 and p65 co-immunoprecipitated with Ash. All these results suggest that Ash may function as a regulator of synaptic vesicle transport through dynamin, p150 and p65.

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Mutational Analysis of the Caenorhabditis elegans Cell-Death Gene ced-3

Genetics ◽

10.1093/genetics/153.4.1655 ◽

1999 ◽

Vol 153 (4) ◽

pp. 1655-1671

Author(s):

Shai Shaham ◽

Peter W Reddien ◽

Brian Davies ◽

H Robert Horvitz

Keyword(s):

Cell Death ◽

Caenorhabditis Elegans ◽

Programmed Cell Death ◽

Mutational Analysis ◽

Acid Sites ◽

Site Directed Mutagenesis ◽

Missense Mutations ◽

Active Site Cysteine ◽

Related Proteins

Abstract Mutations in the gene ced-3, which encodes a protease similar to interleukin-1β converting enzyme and related proteins termed caspases, prevent programmed cell death in the nematode Caenorhabditis elegans. We used site-directed mutagenesis to demonstrate that both the presumptive active-site cysteine of the CED-3 protease and the aspartate residues at sites of processing of the CED-3 proprotein are required for programmed cell death in vivo. We characterized the phenotypes caused by and the molecular lesions of 52 ced-3 alleles. These alleles can be ordered in a graded phenotypic series. Of the 30 amino acid sites altered by ced-3 missense mutations, 29 are conserved with at least one other caspase, suggesting that these residues define sites important for the functions of all caspases. Animals homozygous for the ced-3(n2452) allele, which is deleted for the region of the ced-3 gene that encodes the protease domain, seemed to be incompletely blocked in programmed cell death, suggesting that some programmed cell death can occur independently of CED-3 protease activity.

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

Molecular and Cellular Biology ◽

10.1128/mcb.14.5.2883-2894.1994 ◽

1994 ◽

Vol 14 (5) ◽

pp. 2883-2894

Author(s):

B J Mayer ◽

D Baltimore

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Kinases ◽

Sh3 Domain ◽

Sh2 Domain ◽

Sh3 Domains ◽

Sh2 Domains ◽

Abl Tyrosine Kinase ◽

Transforming Activity

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.

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A conserved proline-rich region of the Saccharomyces cerevisiae cyclase-associated protein binds SH3 domains and modulates cytoskeletal localization.

Molecular and Cellular Biology ◽

10.1128/mcb.16.2.548 ◽

1996 ◽

Vol 16 (2) ◽

pp. 548-556 ◽

Cited By ~ 90

Author(s):

N L Freeman ◽

T Lila ◽

K A Mintzer ◽

Z Chen ◽

A J Pahk ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Adenylyl Cyclase ◽

Sh3 Domain ◽

Regulatory Function ◽

Nucleotide Exchange ◽

Cortical Actin ◽

Rich Region ◽

Sh3 Domains ◽

Proline Rich Region

Saccharomyces cerevisiae cyclase-associated protein (CAP or Srv2p) is multifunctional. The N-terminal third of CAP binds to adenylyl cyclase and has been implicated in adenylyl cyclase activation in vivo. The widely conserved C-terminal domain of CAP binds to monomeric actin and serves an important cytoskeletal regulatory function in vivo. In addition, all CAP homologs contain a centrally located proline-rich region which has no previously identified function. Recently, SH3 (Src homology 3) domains were shown to bind to proline-rich regions of proteins. Here we report that the proline-rich region of CAP is recognized by the SH3 domains of several proteins, including the yeast actin-associated protein Abp1p. Immunolocalization experiments demonstrate that CAP colocalizes with cortical actin-containing structures in vivo and that a region of CAP containing the SH3 domain binding site is required for this localization. We also demonstrate that the SH3 domain of yeast Abp1p and that of the yeast RAS protein guanine nucleotide exchange factor Cdc25p complex with adenylyl cyclase in vitro. Interestingly, the binding of the Cdc25p SH3 domain is not mediated by CAP and therefore may involve direct binding to adenylyl cyclase or to an unidentified protein which complexes with adenylyl cyclase. We also found that CAP homologous from Schizosaccharomyces pombe and humans bind SH3 domains. The human protein binds most strongly to the SH3 domain from the abl proto-oncogene. These observations identify CAP as an SH3 domain-binding protein and suggest that CAP mediates interactions between SH3 domain proteins and monomeric actin.

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Carbodiimide as a tissue fixative in histamine immunohistochemistry and its application in developmental neurobiology.

Journal of Histochemistry & Cytochemistry ◽

10.1177/36.3.3343510 ◽

1988 ◽

Vol 36 (3) ◽

pp. 259-269 ◽

Cited By ~ 144

Author(s):

P Panula ◽

O Häppölä ◽

M S Airaksinen ◽

S Auvinen ◽

A Virkamäki

Keyword(s):

Rat Brain ◽

Nerve Fibers ◽

Immunohistochemical Method ◽

Dot Blot ◽

Culture Dish ◽

Long Fibers ◽

Developing Rat ◽

Strong Immunoreactivity

The object of this study was to develop an immunohistochemical method that could be used to study neuronal histamine, especially in nerve fibers and terminals where most previous methods have not been applicable. Three new antisera were produced in rabbits against conjugated histamine, and the fixative used in conjugation, 1-ethyl-3(3-diamethylaminopropyl)-carbodiimide (EDCDI), was used in tissue fixation and compared to paraformaldehyde. Specificity of the antisera was established with dot-blot tests on nitrocellulose, with blocking controls and affinity-purified antibodies. EDCDI appeared to be superior to paraformaldehyde as a fixative, and histamine-immunoreactive nerve cells were visualized in developing rat brain during late fetal development from embryonal day 12. By the second postnatal week, the distribution of histamine-immunoreactive neurons in rat brain had reached the adult pattern and immunoreactive nerve fibers were seen in many areas. Posterior hypothalamic neurons from newborn rat in vitro showed strong immunoreactivity for histamine and developed long varicose fibers, which covered the culture dish by the end of the fourth week in vitro. Fixation with EDCDI also allowed detection of histamine in gastric enterochromaffin-like cells and mast cells in rat. The results suggest that the histamine-containing neuron system in rat brain develops during the late fetal and early postnatal periods, and that immunoreactive neurons develop long fibers both in vivo and in vitro.

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