The conserved carboxy-terminus of the MscS mechanosensitive channel is not essential but increases stability and activity

The Escherichia coli MscS mechanosensitive channel protein has a distinct domain structure that terminates in a conserved seven-strand beta barrel. This distinctive feature suggested it could be a critical determinant of channel stability and activity. Measurements on a protein deleted for the base of the vestibule and the beta barrel (residues 266-286) suggested that the modified channel had reduced activity. However, induction of the mutant protein resulted in membrane protein accumulation equivalent to wild type and a physiologically functional channel. In patch clamp analysis the activity profile was similar to wild type but reduced numbers of channel were seen per patch, suggesting reduced assembly or stability of the mutant protein. The mutant channel exhibited a subtle change in character - channels did not re-open after full desensitization. Thus the immediate carboxy-terminus (residues 266-286) is not essential for MscS gating but improves stability and activity and is required for recovery of channel activity after desensitization.     

Regulation of human Cdc25A stability by Serine 75 phosphorylation is not sufficient to activate a S phase checkpoint

Degradation of Cdc25A phosphatase is an ubiquitous feature of stress. There are some discrepancies in the reported roles for different phosphorylation sites in the regulation of Cdc25A stability. Using a panel of doxycycline-inducible phosphorylation mutants we show that the stability of human Cdc25A protein is dependent upon phosphorylation at S75. In non-stressed conditions and in non-mitotic cells, Cdc25A is unstable and its stability is regulated in a Chk1-dependent manner. During mitosis, Cdc25A becomes stable and does not undergo degradation after DNA damage. We further show that Chk1 kinase regulates Cdc25A stability after UV irradiation. Similar to Chk1 kinase, p38 MAPK controls Cdc25A protein level after osmotic stress. Using phospho-specific antibodies, we find that both kinases can phosphorylate S75 and S123 in vitro. Inactivation of either Chk1 after UV-irradiation or p38 MAPK after osmotic stress prevents activation of a S phase checkpoint and S75 and S123 phosphorylation. However, introduction of stable Cdc25A (S75A or S75/123A) proteins is not sufficient to overcome this checkpoint. We propose that regulation of human Cdc25A stability by its phosphorylation at S75 may contribute to S phase checkpoint activation only in cooperation with other regulatory mechanisms.     

The reduced stability of a plant alcohol dehydrogenase is due to the substitution of serine for a highly conserved phenylalanine residue

The zinc-binding long-chain alcohol dehydrogenases from plants and animals exhibit a considerable level of amino acid sequence conservation. While the functional importance of many of the conserved residues is known, the role of others has not yet been determined. We have identified a naturally occurring Adh-1 allele in the legume Phaseolus acutifolius with several unusual characteristics. Individuals homozygous for this allele, Adh-1CN, possess a single isozyme starch gel electrophoretic pattern suggestive of a null allele, and exhibit ADH enzyme activity levels ca. 60% lower than the standard wild-type Adh-1F line. Interestingly, analysis of Adh-1CN homozygotes on an alternative gel system indicates that Adh-1CN does encode a polypeptide capable of forming functional homo- and heterodimers. However, the levels of ADH activity displayed by these isozymes are far lower than those observed for the corresponding wild type ADH-1F isozymes. Dialysis experiments indicate that isozymes containing the ADH-1CN polypeptide are inactivated by slightly acidic conditions, which may explain the apparent null phenotype on starch gels. Elevated temperatures cause a similar loss of enzyme activity. The deduced amino acid sequences of ADH-1CN and ADH-1F were obtained from their corresponding cDNA clones, and the only significant difference detected between the two is a single amino acid replacement substitution. Residue 144 is occupied by phenylalanine in the ADH-1F polypeptide, whereas serine occupies this position in the ADH-1CN polypeptide. The proximity of residue 144 to the catalytic zinc in the substrate-binding pocket, coupled with the fact that it is integral to a defined hydrophobic core of the ADH polypeptide, may explain the observed disruptive effect that the serine substitution has on both the activity and stability of the ADH-1CN polypeptide. It also provides an explanation for the maintenance of phenylalanine or the structurally similar tyrosine at this residue in Zn-binding long-chain ADHs.     

The molecular chaperones Hsp90 and Hsc70 are both necessary and sufficient to activate hormone binding by glucocorticoid receptor

Glucocorticoid receptors must be complexed with Hsp90 in order to bind steroids, and it has been reported that at least three other proteins, Hop, Hsc70, and a J-domain protein (either Hsp40 or Ydj1), are required for formation of active Hsp90-steroid receptor complex. In the present study, we reinvestigated activation of stripped steroid receptors isolated from either L cells or WCL2 cells. Surprisingly, we found, using highly purified proteins, that only Hsp90 and Hsc70 are required for the activation of glucocorticoid receptors in the presence of steroids; in the absence of steroids, either p23 or molybdate are also required as reported previously. Addition of Hop or Ydj1 had no affect on the rate or magnitude of the activation of the stripped receptors, and quantitative Western blots confirmed that neither Hop or Hsp40 were present in our protein preparations or in the stripped receptors. Furthermore, a truncated recombinant Hsp70 that does not bind Hop or Hsp40 was as effective as wild-type Hsp70 in activating stripped receptor. Since Hsc70 does not bind directly to Hsp90 but both proteins bind to Hop, it has been suggested that Hop acts as a bridge between Hsp90 and Hsp70. However, we found that after Hsc70 or Hsp90 bind directly to the stripped receptors, they are fully reactivated by Hsp90 or Hsc70, respectively. We, therefore, conclude that Hsp90 and Hsc70 bind independently to stripped glucocorticoid receptors and alone are sufficient to activate them to bind steroids.     

The C-terminal region of OsWRKY30 is sufficient to confer enhanced resistance to pathogen and activate the expression of defense-related genes

WRKY proteins are known to play major roles in defense signaling. We identified a pathogen-inducible and SA-inducible OsWRKY30. Our cDNA clone encodes the C-terminal region (CTR) of OsWRKY30. CTR-OsWRKY30 includes the C-terminal WRKY domain and nuclear localization sequence. CTR-OsWRKY30 was sufficient to bind W-box sequences (TTGACC/T). Over-expression of the CTR-OsWRKY30 resulted in enhanced resistance to pathogens in Arabidopsis and rice. Defense-related genes were constitutively expressed in transgenic Arabidopsis and rice over-expressing CTR-OsWRKY30. Based on promoter transient assays, CTR-OsWRKY30 is sufficient to activate OsPR10a promoter as much as full length OsWRKY30. Taken together, CTR-OsWRKY30 positively regulates defense signaling, thereby resulting in enhanced resistance to pathogens.     

Colocalization of sensors is sufficient to activate the DNA damage checkpoint in the absence of damage

Previous work on the DNA damage checkpoint in Saccharomyces cerevisiae has shown that two complexes independently sense DNA lesions: the kinase Mec1-Ddc2 and the PCNA-like 9-1-1 complex. To test whether colocalization of these components is sufficient for checkpoint activation, we fused these checkpoint proteins to the LacI repressor and artificially colocalized these fusions by expressing them in cells harboring Lac operator arrays. We observed Rad53 and Rad9 phosphorylation, Sml1 degradation, and metaphase delay, demonstrating that colocalization of these sensors is sufficient to activate the checkpoint in the absence of DNA damage. Our tethering system allowed us to establish that CDK functions in the checkpoint pathway downstream of damage processing and checkpoint protein recruitment. This CDK dependence is likely, at least in part, through Rad9, since mutation of CDK consensus sites compromised its checkpoint function.     

Mutual antagonism between signals secreted by adjacent wingless and engrailed cells leads to specification of complementary regions of the Drosophila parasegment.

Specialized groups of cells known as organizers govern the establishment of cell type diversity across cellular fields. Segmental patterning within the Drosophila embryonic epidermis is one paradigm for organizer function. Here cells differentiate into smooth cuticle or distinct denticle types. At parasegment boundaries, cells expressing Wingless confront cells co-expressing Engrailed and Hedgehog. While Wingless is essential for smooth cell fates, the signals that establish denticle diversity are unknown. We show that wg mutants have residual mirror-symmetric pattern that is due to an Engrailed-dependent signal specifying anterior denticle fates. The Engrailed-dependent signal acts unidirectionally and Wg activity imposes this asymmetry. Reciprocally, the Engrailed/Hedgehog interface imposes asymmetry on Wg signaling. Thus, a bipartite organizer, with each signal acting essentially unidirectionally, specifies segmental pattern.     

Coordinated signaling through both G12/13 and G(i) pathways is sufficient to activate GPIIb/IIIa in human platelets

Activation of GPIIb/IIIa is known to require agonist-induced inside-out signaling through G(q), G(i), and G(z). Although activated by several platelet agonists, including thrombin and thromboxane A(2), the contribution of the G(12/13) signaling pathway to GPIIb/IIIa activation has not been investigated. In this study, we used selective stimulation of G protein pathways to investigate the contribution of G(12/13) activation to platelet fibrinogen receptor activation. YFLLRNP is a PAR-1-specific partial agonist that, at low concentrations (60 microm), selectively activates the G(12/13) signaling cascade resulting in platelet shape change without stimulating the G(q) or G(i) signaling pathways. YFLLRNP-mediated shape change was completely inhibited by the p160(ROCK) inhibitor, Y-27632. At this low concentration, YFLLRNP-mediated G(12/13) signaling caused platelet aggregation and enhanced PAC-1 binding when combined with selective G(i) or G(z) signaling, via selective stimulation of the P2Y(12) receptor or alpha(2A)-adrenergic receptor, respectively. Similar data were obtained when using low dose (10 nm), a thromboxane A(2) mimetic, to activate G(12/13) in the presence of G(i) signaling. These results suggest that selective activation of G(12/13) causes platelet GPIIb/IIIa activation when combined with G(i) signaling. Unlike either G(12/13) or G(i) activation alone, co-activation of both G(12/13) and G(i) resulted in a small increase in intracellular calcium. Chelation of intracellular calcium with dimethyl BAPTA dramatically blocked G(12/13) and G(i)-mediated platelet aggregation. No significant effect on aggregation was seen when using selective inhibitors for p160(ROCK), PKC, or MEKK1. PI 3-kinase inhibition lead to near abolishment of platelet aggregation induced by co-stimulation of G(q) and G(i) pathways, but not by G(12/13) and G(i) pathways. These data demonstrate that co-stimulation of G(12/13) and G(i) pathways is sufficient to activate GPIIb/IIIa in human platelets in a mechanism that involves intracellular calcium, and that PI 3-kinase is an important signaling molecule downstream of G(q) but not downstream of G(12/13) pathway.     

The vinculin binding sites of talin and alpha-actinin are sufficient to activate vinculin

Vinculin regulates both cell-cell and cell-matrix junctions and anchors adhesion complexes to the actin cytoskeleton through its interactions with the vinculin binding sites of alpha-actinin or talin. Activation of vinculin requires a severing of the intramolecular interactions between its N- and C-terminal domains, which is necessary for vinculin to bind to F-actin; yet how this occurs in cells is not resolved. We tested the hypothesis that talin and alpha-actinin activate vinculin through their vinculin binding sites. Indeed, we show that these vinculin binding sites have a high affinity for full-length vinculin, are sufficient to sever the head-tail interactions of vinculin, and they induce conformational changes that allow vinculin to bind to F-actin. Finally, microinjection of these vinculin binding sites specifically targets vinculin in cells, disrupting its interactions with talin and alpha-actinin and disassembling focal adhesions. In their native (inactive) states the vinculin binding sites of talin and alpha-actinin are buried within helical bundles present in their central rod domains. Collectively, these results support a model where the engagement of adhesion receptors first activates talin or alpha-actinin, by provoking structural changes that allow their vinculin binding sites to swing out, which are then sufficient to bind to and activate vinculin.     

The evolutionarily conserved N-terminal region of Cbl is sufficient to enhance down-regulation of the epidermal growth factor receptor

The mammalian proto-oncoprotein Cbl and its homologues in Caenorhabditis elegans and Drosophila are evolutionarily conserved negative regulators of the epidermal growth factor receptor (EGF-R). Overexpression of wild-type Cbl enhances down-regulation of activated EGF-R from the cell surface. We report that the Cbl tyrosine kinase-binding (TKB) domain is essential for this activity. Whereas wild-type Cbl enhanced ligand-dependent EGF-R ubiquitination, down-regulation from the cell surface, accumulation in intracellular vesicles, and degradation, a Cbl TKB domain-inactivated mutant (G306E) did not. Furthermore, the transforming truncation mutant Cbl-N (residues 1-357), comprising only the Cbl TKB domain, functioned as a dominant negative protein. It colocalized with EGF-R in intracellular vesicular structures, yet it suppressed down-regulation of EGF-R from the surface of cells expressing endogenous wild-type Cbl. Therefore, Cbl-mediated down-regulation of EGF-R requires the integrity of both the N-terminal TKB domain and additional C-terminal sequences. A Cbl truncation mutant comprising amino acids 1-440 functioned like wild-type Cbl in down-regulation assays. This mutant includes the evolutionarily conserved TKB and RING finger domains but lacks the less conserved C-terminal sequences. We conclude that the evolutionarily conserved N terminus of Cbl is sufficient to effect enhancement of EGF-R ubiquitination and down-regulation from the cell surface.