Identification of novel point mutations in ERK2 that selectively disrupt binding to MEK1

Extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2) are essential components of pathways through which signals received at membrane receptors are converted into specific changes in protein function and gene expression. As with other members of the mitogen-activated protein (MAP) kinase family, ERK1 and ERK2 are activated by phosphorylations catalyzed by dual-specificity protein kinases known as MAP/ERK kinases (MEKs). MEKs exhibit stringent specificity for individual MAP kinases. Indeed, MEK1 and MEK2 are the only known activators of ERK1 and ERK2. ERK2 small middle dotMEK1/2 complexes can be detected in vitro and in vivo. The biochemical nature of such complexes and their role in MAP kinase signaling are under investigation. This report describes the use of a yeast two-hybrid screen to identify point mutations in ERK2 that impair its interaction with MEK1/2, yet do not alter its interactions with other proteins. ERK2 residues identified in this screen are on the surface of the C-terminal domain of the kinase, either within or immediately preceding alpha-helix G, or within the MAP kinase insert. Some mutations identified in this manner impaired the two-hybrid interaction of ERK2 with both MEK1 and MEK2, whereas others had a predominant effect on the interaction with either MEK1 or MEK2. Mutant ERK2 proteins displayed reduced activation in HEK293 cells following epidermal growth factor treatment, consistent with their impaired interaction with MEK1/2. However, ERK2 proteins containing MEK-specific mutations retained kinase activity, and were similar to wild type ERK2 in their activation following overexpression of constitutively active MEK1. Unlike wild type ERK2, proteins containing MEK-specific point mutations were constitutively localized in the nucleus, even in the presence of overexpressed MEK1. These data suggest an essential role for the MAP kinase insert and residues within or just preceding alpha-helix G in the interaction of ERK2 with MEK1/2.     

The immunomodulatory protein B7-H4 is overexpressed in breast and ovarian cancers and promotes epithelial cell transformation

B7-H4 protein is expressed on the surface of a variety of immune cells and functions as a negative regulator of T cell responses. We independently identified B7-H4 (DD-O110) through a genomic effort to discover genes upregulated in tumors and here we describe a new functional role for B7-H4 protein in cancer. We show that B7-H4 mRNA and protein are overexpressed in human serous ovarian cancers and breast cancers with relatively little or no expression in normal tissues. B7-H4 protein is extensively glycosylated and displayed on the surface of tumor cells and we provide the first demonstration of a direct role for B7-H4 in promoting malignant transformation of epithelial cells. Overexpression of B7-H4 in a human ovarian cancer cell line with little endogenous B7-H4 expression increased tumor formation in SCID mice. Whereas overexpression of B7-H4 protected epithelial cells from anoikis, siRNA-mediated knockdown of B7-H4 mRNA and protein expression in a breast cancer cell line increased caspase activity and apoptosis. The restricted normal tissue distribution of B7-H4, its overexpression in a majority of breast and ovarian cancers and functional activity in transformation validate this cell surface protein as a new target for therapeutic intervention. A therapeutic antibody strategy aimed at B7-H4 could offer an exciting opportunity to inhibit the growth and progression of human ovarian and breast cancers.     

Molecular Marker Based Coefficient of Parentage Analysis for Establishing Distinctness in Indian Rice (Oryza sativa L) Varieties

Molecular characterization of 32 Indian rice varieties of different agro-climatic zones resulted in mean heterozygosity value of 0.622, 0.819 and 0.890 over polymorphic loci and marker index value of 1.00, 6.75 and 4.16, respectively for RAPD, ISSR and STMS primers. The three marker systems resulted in 201 polymorphic bands (94.36%) out of a total of 213 bands. The probability of a chance identical match between two varieties was very low (2.08x10¹⁰) in combined molecular marker analysis as compared to individual marker system (RAPD, 7.5 x 10^-4; ISSR, 1.5 x 10^-3 and STMS, 3.9 x 10^-6). The combined average genetic similarity for molecular markers and coefficient of parentage (COP) across all 496 pairwise combinations revealed a non-significant relationship (r = 0.215).     

Social Factors and Leukocyte DNA Methylation of Repetitive Sequences: The Multi-Ethnic Study of Atherosclerosis

Epigenetic changes are a potential mechanism contributing to race/ethnic and socioeconomic disparities in health. However, there is scant evidence of the race/ethnic and socioeconomic patterning of epigenetic marks. We used data from the Multi-Ethnic Study of Atherosclerosis Stress Study (N = 988) to describe age- and gender- independent associations of race/ethnicity and socioeconomic status (SES) with methylation of Alu and LINE-1 repetitive elements in leukocyte DNA. Mean Alu and Line 1 methylation in the full sample were 24% and 81% respectively. In multivariable linear regression models, African-Americans had 0.27% (p<0.01) and Hispanics 0.20% (p<0.05) lower Alu methylation than whites. In contrast, African-Americans had 0.41% (p<0.01) and Hispanics 0.39% (p<0.01) higher LINE-1 methylation than whites. These associations remained after adjustment for SES. In addition, a one standard deviation higher wealth was associated with 0.09% (p<0.01) higher Alu and 0.15% (p<0.01) lower LINE-1 methylation in age- and gender- adjusted models. Additional adjustment for race/ethnicity did not alter this pattern. No associations were observed with income, education or childhood SES. Our findings, from a large community-based sample, suggest that DNA methylation is socially patterned. Future research, including studies of gene-specific methylation, is needed to understand better the opposing associations of Alu and LINE-1 methylation with race/ethnicity and wealth as well as the extent to which small methylation changes in these sequences may influence disparities in health.     

Pathogen Exposure in Cattle at the Livestock-Wildlife Interface

Land use is an important driver of variation in human infectious disease risk, but less is known about how land use affects disease risk in livestock. To understand how land use is associated with disease risk in livestock, we examined patterns of pathogen exposure in cattle across two livestock ranching systems in rural Kenya: private ranches with low- to medium-intensity cattle production and high wildlife densities, and group ranches with high-intensity cattle production and low wildlife densities. We surveyed cattle from six ranches for three pathogens: Brucella spp., bovine viral diarrhea virus (BVDV) and Leptospira serovar Hardjo. We found that exposure risk for Leptospira was higher on private ranches than on group ranches, but there was no difference in exposure by ranch type for Brucella or BVDV. We hypothesize that variation in livestock and wildlife contact patterns between ranch types may be driving the pattern observed for Leptospira exposure and that the different relationships we found between exposure risk and ranch type by pathogen may be explained by differences in transmission mode. Overall, our results suggest that wildlife–livestock contact patterns may play a key role in shaping pathogen transmission to livestock and that the magnitude of such effects likely depend on characteristics of the pathogen in question.     

TAO (thousand-and-one amino acid) protein kinases mediate signaling from carbachol to p38 mitogen-activated protein kinase and ternary complex factors

The TAO (for thousand-and-one amino acids) protein kinases activate p38 mitogen-activated protein (MAP) kinase cascades in vitro and in cells by phosphorylating the MAP/ERK kinases (MEKs) 3 and 6. We found that TAO2 activity was increased by carbachol and that carbachol and the heterotrimeric G protein Galphao could activate p38 in 293 cells. Using dominant interfering kinase mutants, we found that MEKs 3 and 6 and TAOs were required for p38 activation by carbachol or the constitutively active mutant GalphaoQ205L. To explore events downstream of TAOs, the effects of TAO2 on ternary complex factors (TCFs) were investigated. Transfection studies demonstrated that TAO2 stimulates phosphorylation of the TCF Elk1 on the major activating site, Ser383, and that TAO2 stimulates transactivation of Elk1 and the related TCF, Sap1. Reporter activity was reduced by the p38-selective inhibitor SB203580. Taken together, these studies suggest that TAO protein kinases relay signals from carbachol through heterotrimeric G proteins to the p38 MAP kinase, which then activates TCFs in the nucleus.