Molecular characterization of the genomes of actinophages SH3, SH10, SH11, and SH12 infecting Streptomyces hygroscopicus.

Summary Some physico-chemical properties of the DNAs released from the actinophages SH3, SH10, SH11, and SH12 are described. The four phage DNAs have a linear double-stranded secondary structure and are unique with respect to their high G·C contents which, from melting studies and buoyant density experiments, were found to be in the range of 68–73 mol-%. The DNA molecular weights were determined by sedimentation velocity experiments and by electron microscopic length measurements, the mean values of the two corresponding data sets being 34.0·10⁶ (SH3), 26.7·10⁶ (SH10), 26.1·10⁶ (SH11), and 28.7·10⁶ (SH12) with a mean relative error of ±5%. From different observations it was concluded that SH10 DNA, and possibly also SH11 and SH12 DNA, have cohesive ends and can undergo intramolecular or intermolecular association to form ring-like monomers or linear and ring-like multimers. Cleavage of the DNAs of SH3, SH10, SH11, and SH12 by EcoRI restriction endonuclease delivered two, one, zero, and two cleavage sites, respectively, and by BamHI restriction endonuclease eight, zero, zero, and zero cleavage sites, respectively.     

Restriction endonuclease analysis of DNA from the Streptomyces phages SH3, SH5, SH10 and SH13

Using 26 restriction endonucleases, a cleavage site survey was undertaken for DNAs of several unrelated Streptomyces phages SH3, SH5, SH10 and SH13. Only EcoRI was found to produce single cleavage in SH3 and SH10 DNA. The complete maps were prepared for the 2, 9 and 11 fragments of SH10 DNA, as generated by EcoRI, KpnI and BglII, respectively. The evidence is presented that SH10 DNA contains cohesive ends. Moreover, a clearplaque mutant of SH10 was shown to contain a deletion of 790 bp in the right part of the genome, including two KpnI sites.     

SH3GLB, a new endophilin-related protein family featuring an SH3 domain

A new cDNA encoding a protein of 362 amino acids designated SH3GLB1, for SH3 domain GRB2-like endophilin B1, was identified in a yeast two-hybrid screen devoted to the identification of new partners interacting with the apoptosis inducer Bax. SH3GLB1 shows strong similarities to the SH3 domain-containing proteins of the endophilin family and presumably represents the human homologue of the potential Caenorhabditis elegans SH3 containing-protein identified by systematic translation of the C. elegans genome (GenBank Accession No. U46675). Reversing prey to bait in the yeast screen, a second protein, SH3GLB2, of 395 amino acids showing 65% identity to SH3GLB1 was identified as an interacting partner of SH3GLB1. The discovery of SH3GLB1 itself in the screening with SH3GLB1 as a bait and further mapping experiments demonstrated that a core coiled-coil-type region is required for the formation of SH3GLB homo- and/or heterodimers, whereas the SH3 domain is not involved in these interactions. Interestingly, the similarities with the endophilin proteins cover the entire sequence of the SH3GLB family, suggesting a common fold and presumably a common mode of action. Furthermore, SH3GLB members colocalize to the cytoplasmic compartment of the cell together with Bax and are excluded from the nucleus. SH3GLB1 and SH3GLB2 do not significantly influence the onset and time course of Bax-mediated apoptosis in HeLa or 293T cells.     

Signalling through SH2 and SH3 domains

In 1986, Pawson's group recognized a region of homology between two oncogenic tyrosine kinases that lay outside the catalytic domain. They termed this the Src homology 2, or SH2, domain. In the ensuing years, SH2 domains have been found in an impressive variety of proteins, as has a second region of homology, inevitably termed SH3. These domains appear to mediate controlled protein-protein interactions. Many proteins that contain SH2 and SH3 domains are involved in signal transduction, suggesting a new paradigm for regulation of intracellular signalling pathways.     

Identification of Src, Fyn, and Lyn SH3-binding proteins: implications for a function of SH3 domains.

Src homology 3 (SH3) domains mediate protein-protein interactions necessary for the coupling of cellular proteins involved in intracellular signal transduction. We previously established solution-binding conditions that allow affinity isolation of Src SH3-binding proteins from cellular extracts (Z. Weng, J. A. Taylor, C. E. Turner, J. S. Brugge, and C. Seidel-Dugan, J. Biol. Chem. 268:14956-14963, 1993). In this report, we identified three of these proteins: Shc, a signaling protein that couples membrane tyrosine kinases with Ras; p62, a protein which can bind to p21rasGAP; and heterogeneous nuclear ribonucleoprotein K, a pre-mRNA-binding protein. All of these proteins contain proline-rich peptide motifs that could serve as SH3 domain ligands, and the binding of these proteins to the Src SH3 domain was inhibited with a proline-rich Src SH3 peptide ligand. These three proteins, as well as most of the other Src SH3 ligands, also bound to the SH3 domains of the closely related protein tyrosine kinases Fyn and Lyn. However, Src- and Lyn-specific SH3-binding proteins were also detected, suggesting subtle differences in the binding specificity of the SH3 domains from these related proteins. Several Src SH3-binding proteins were phosphorylated in Src-transformed cells. The phosphorylation of these proteins was not detected in cells transformed by a mutant variant of Src lacking the SH3 domain, while there was little change in tyrosine phosphorylation of other Src-induced phosphoproteins. In addition, the coprecipitation of v-Src with two tyrosyl-phosphorylated proteins with M(r)s of 62,000 and 130,000 was inhibited by incubation with a Src SH3 peptide ligand, suggesting that the binding of these substrate proteins is dependent on interactions with the SH3 domain. These results strongly suggest a role for the Src SH3 domain in the recruitment of substrates to this protein tyrosine kinase, either through direct interaction with the SH3 domain or indirectly through interactions with proteins that bind to the SH3 domain.     

Geology of Archeological Site 48SH312, Wyoming

Abstract

Archeological Site 48SH312 occurs upon slightly weathered shale in the uppermost portion ot the Fort Union Formation (Paleocene) of eastern Sheridan County, Wyoming. Only a trace of a paleosol is developed on the shale. Colluvium overlies the shale. The site is a small remnant of a ravine floor which existed during Kaycee time. Physiography of the present ravine and local distribution of resistant lenticular strata in the Fort Union Formation, suggest that the paleo-ravine was a box ravine with near vertical walls of 5-10 meters. Paleosol development, weathering features, and homogeneity of colluvium are interpreted as indicating a surface that represents late Kaycee time and provides a geological age for the site of 3500 to 4000 years before present.     

A new, smaller actin-activatable myosin subfragment 1 which lacks the 20-kDa, SH1 and SH2 peptide

The actin-dependent ATPase activity of myosin is retained in the separated heads (S1) which contain the NH2-terminal 95-kDa heavy chain fragment and one or two light chains. The S1 heavy chain can be degraded further by limited trypsin treatment into characteristic 25-, 50-, and 20-kDa peptides, in this order from the NH2-terminal end. The 20-kDa peptide contains an actin-binding site and SH1 and SH2, two thiols whose modification dramatically affects ATPase activity. By treating myosin filaments with trypsin at 4 degrees C in the presence of 2 mM MgCl2, we have now obtained preferential cleavage at the 50-20-kDa heavy chain site without any cleavage at the head-rod junction and hinge region in the rod. Incubation of these trypsinized filaments at 37 degrees C in the presence of MgATP released a new S1 fraction which lacked the COOH-terminal 20-kDa heavy chain peptide region. This fraction, termed S1'(75K), has more than 50% of the actin-activated Mg2+-ATPase activity of S1 and the characteristic Ca2+-ATPase and K+-EDTA ATPase activities of myosin. These results show that SH1 and SH2 are not essential for ATPase activity and that binding of actin to the 20-kDa region is not essential for the enhancement of the Mg2+-ATPase activity.     

The kinase, SH3, and SH2 domains of Lck play critical roles in T-cell activation after ZAP-70 membrane localization.

Antigenic stimulation of the T-cell antigen receptor initiates signal transduction through the immunoreceptor tyrosine-based activation motifs (ITAMs). When its two tyrosines are phosphorylated, ITAM forms a binding site for ZAP-70, one of the cytoplasmic protein tyrosine kinases essential for T-cell activation. The signaling process that follows ZAP-70 binding to ITAM has been analyzed by the construction of fusion proteins that localize ZAP-70 to the plasma membrane. We found that membrane-localized forms of ZAP-70 induce late signaling events such as activation of nuclear factor of activated T cells without any stimulation. This activity was observed only when Lck was expressed and functional. In addition, each mutation that affects the function of Lck in the kinase, Src homology 2 (SH2), and SH3 domains greatly impaired the signaling ability of the chimeric protein. Therefore, Lck functions in multiple manners in T-cell activation for the steps following ZAP-70 binding to ITAM.     

SH2 domains, interaction modules and cellular wiring

SH2 domains serve as the prototype for a growing family of protein-interaction modules, characteristic of polypeptides involved in transmitting signals from external and internal cues. The specific interactions of proteins with one another, and with other cellular components such as phospholipids and nucleic acids, provide a very general device to organize cellular behavior. We discuss the idea that rewiring of the cell's interaction network by pathogenic microorganisms and mutant cellular proteins contributes to dysregulation of cell signaling and thus to disease.     

Tyrosine phosphorylation of tau regulates its interactions with Fyn SH2 domains, but not SH3 domains, altering the cellular localization of tau

Recent reports have demonstrated that interactions between the microtubule-associated protein tau and the nonreceptor tyrosine kinase Fyn play a critical role in mediating synaptic toxicity and neuronal loss in response to β-amyloid (Aβ) in models of Alzheimer's disease. Disruption of interactions between Fyn and tau may thus have the potential to protect neurons from Aβ-induced neurotoxicity. Here, we investigated tau and Fyn interactions and the potential implications for positioning of these proteins in membrane microdomains. Tau is known to bind to Fyn via its Src-homology (SH)3 domain, an association regulated by phosphorylation of PXXP motifs in tau. Here, we show that Pro216 within the PXXP(213-216) motif in tau plays an important role in mediating the interaction of tau with Fyn-SH3. We also show that tau interacts with the SH2 domain of Fyn, and that this association, unlike that of Fyn-SH3, is influenced by Fyn-mediated tyrosine phosphorylation of tau. In particular, phosphorylation of tau at Tyr18, a reported target of Fyn, is important for mediating Fyn-SH2-tau interactions. Finally, we show that tyrosine phosphorylation influences the localization of tau to detergent-resistant membrane microdomains in primary cortical neurons, and that this trafficking is Fyn-dependent. These findings may have implications for the development of novel therapeutic strategies aimed at disrupting the tau/Fyn-mediated synaptic dysfunction that occurs in response to elevated Aβ levels in neurodegenerative disease.     

Membrane-targeting of signalling molecules by SH2/SH3 domain-containing adaptor proteins

SH2/SH3 domain-containing adaptor proteins play a critical role in regulating tyrosine kinase signalling pathways. The major function of these adaptors, such as Grb2, Nck, and Crk, is to recruit proline-rich effector molecules to tyrosine-phosphorylated kinases or their substrates. In recent years dozens of novel proteins have emerged that are capable of associating with the SH2 and the SH3 domains of adaptors. In this review, the author attempts to summarise these novel binding partners of Grb2, Nck, and Crk, and to discuss current controversies regarding function and regulation of protein multicomplexes held together by SH2/SH3 adaptor molecules at the plasma membrane.     

Surface Loops in a Single SH2 Domain Are Capable of Encoding the Spectrum of Specificity of the SH2 Family,

1. Download : Download high-res image (119KB)
2. Download : Download full-size image

Highlights

• •Surface loops play an essential role in SH2 domain specificity.
• •Diverse specificities may be obtained from a single SH2 domain by combinatorial mutations in the EF and BG loops.
• •The specificity of a loop mutant correlates with the sequence characteristics of the bait peptide used in its isolation.

     

Membrane-targeting of signalling molecules by SH2/SH3 domain-containing adaptor proteins.

SH2/SH3 domain-containing adaptor proteins play a critical role in regulating tyrosine kinase signalling pathways. The major function of these adaptors, such as Grb2, Nck, and Crk, is to recruit proline-rich effector molecules to tyrosine-phosphorylated kinases or their substrates. In recent years dozens of novel proteins have emerged that are capable of associating with the SH2 and the SH3 domains of adaptors. In this review, the author attempts to summarise these novel binding partners of Grb2, Nck, and Crk, and to discuss current controversies regarding function and regulation of protein multicomplexes held together by SH2/SH3 adaptor molecules at the plasma membrane.