Specificity of HCPTP variants toward EphA2 tyrosines by quantitative selected reaction monitoring

EphA2 receptor tyrosine kinase and the human cytoplasmic protein tyrosine phosphatase (HCPTP) are overexpressed in a number of epithelial cancers. Overexpressed EphA2 in these cancers shows a significant decrease in phosphotyrosine content which results in suppression of receptor signaling and endocytosis and an increase in metastatic potential. The decreased phosphotyrosine content of EphA2 has been associated with decreased contact with its ligand, ephrin A1 and dephosphorylation by HCPTP. Potential specificity of the two HCPTP variants for tyrosines on EphA2 has not been investigated. We have used a mass spectrometry assay to measure relative rates of dephosphorylation for the two HCPTP variants at phosphotyrosine sites associated with control of the EphA2 kinase activity or interaction with downstream targets. Our results suggest that although both variants dephosphorylate the EphA2 receptor, the rate and specificity of dephosphorylation for specific tyrosines are different for HCPTP-A and HCPTP-B. The SAM domain tyrosine Y960 which has been implicated in downstream PI3K signaling is dephosphorylated exclusively by HCPTP-B. The activation loop tyrosine (Y772) which directly controls kinase activity is dephosphorylated about six times faster by HCPTP-A. In contrast, the juxtamembrane tyrosines (Y575, Y588 and Y594) which are implicated in both control of kinase activity and downstream signaling are dephosphorylated by both variants with similar rates. This difference in preference for dephosphorylation sites on EphA2 not only illuminates the different roles of the two variants of the phosphatase in EphA2 signaling, but also explains why both HCPTP variants are highly conserved in most mammals

Needle chlorosis in Sitka spruce following a three-year exposure to low concentrations of ozone: changes in mineral content, pigmentation and ascorbic acid.

Two-year-old seedlings of Sitka spruce were exposed to 70 nl 1−1 ozone or to filtered air over three successive summers in outdoor large-scale fumigation chambers (Solardomes). Seven months after the last period of exposure to the pollutant and just prior to budburst, upper-surface chlorisis affecting only the older needles of ozone-exposed trees was observed. In many respects, the symptoms appeared to be similar to those characteristic of type 1 spruce damage occurring in parts of mainland Europe. Chlorophyll pigments were reduced in the ozone-exposed older foliage, but no change in the ratio of chlorophylls to carotene was observed. The content of ascorbic acid was clearly related to the amount of foliar damage observed on the trees exposed to ozone and the largest increases were seen in those trees which were most visibly damaged. Although none of the foliage examined was deficient in any of the nutrient cations which were measured, the concentration of Mg in the older needles was significantly reduced by exposure to ozone, irrespective of damage symptoms. Exposure to ozone also resulted in increases in the ratios of K: Mg and Ca: Mg. In the older needles, leaching of Mg2− and K− by 0.5 mM H2SO4, pH 3.0, was enhanced by prior exposure to ozone, but the amounts removed were small (< 6%). It is suggested that long-term exposure to ozone has a cumulative effect on plant tissue and that the observed chlorosis was the result of accelerated senescence