Priming-dependent phosphorylation and regulation of the tumor suppressor pVHL by glycogen synthase kinase 3.

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is linked to the development of tumors of the eyes, kidneys, and central nervous system. VHL encodes two gene products, pVHL30 and pVHL19, of which one, pVHL30, associates functionally with microtubules (MTs) to regulate their stability. Here we report that pVHL30 is a novel substrate of glycogen synthase kinase 3 (GSK3) in vitro and in vivo. Phosphorylation of pVHL on serine 68 (S68) by GSK3 requires a priming phosphorylation event at serine 72 (S72) mediated in vitro by casein kinase I. Functional analysis of pVHL species carrying nonphosphorylatable or phosphomimicking mutations at S68 and/or S72 reveals a central role for these phosphorylation events in the regulation of pVHL's MT stabilization (but not binding) activity. Taken together, our results identify pVHL as a novel priming-dependent substrate of GSK3 and suggest a dual-kinase mechanism in the control of pVHL's MT stabilization function. Since GSK3 is a component of multiple signaling pathways that are altered in human cancer, our results further imply that normal operation of the GSK3-pVHL axis may be a critical aspect of pVHL's tumor suppressor mechanism through the regulation of MT dynamics.

Enhanced sensitivity of protein kinase B/Akt to insulin in hypoxia is independent of HIF1alpha and promotes cell viability.

Maintenance of oxygen homeostasis is a key requirement to ensure normal mammalian cell growth and differentiation. Hypoxia arises when oxygen demand exceeds supply, and is a feature of multiple human diseases including stroke, cancer and renal fibrosis. We have investigated the effect of hypoxia on kidney cells, and observed that insulin-induced cell viability is increased in hypoxia. We have characterized the role of protein kinase B (PKB/Akt) in these cells as a potential mediator of this effect. PKB/Akt activity was increased by low oxygen concentrations in kidney cells, and insulin-stimulated activation of PKB/Akt was stronger, more rapid and more sustained in hypoxia. Reduction of HIF1alpha levels using antimycin-A or siRNA targeting HIF1alpha did not affect PKB/Akt activation in hypoxia. Pharmacologic stabilization of HIF1alpha independent of hypoxia did not increase insulin-stimulated PKB/Akt activation. Although increased insulin-stimulated cell viability was observed in hypoxia, no differences in the degree of insulin-stimulated glucose uptake were observed in L6 muscle cells in hypoxia compared to normoxia. Thus, PKB/Akt may regulate specific cellular responses to growth factors such as insulin under adverse conditions such as hypoxia.

Impact of dyslipidaemia on arterial structure and function in urban indigenous Australians.

BACKGROUND: Premature cardiovascular disease (CDV) is highly prevalent in urban Indigenous Australians. We studied arterial structure and function in 144 volunteers aged 15-66 years to assess the role of dyslipidaemia and other traditional vascular risk factors on cardiovascular risk in young and older urban Indigenous Australians. METHODS: We assessed carotid intima-media thickness (CIMT) by high-resolution B-mode ultrasound imaging of the common carotid artery and peripheral wave reflection using applanation tonometry to obtain the aortic augmentation index (AI) in Indigenous Australian participants of the Darwin Region Urban Indigenous Diabetes (DRUID) study. RESULTS: Participants aged 15-24 years demonstrated fewer cardiovascular risk factors than the older group (25-66 years) and predictors of CIMT and AI differed between younger and older groups. CIMT was higher in the older group (0.67mm vs. 0.61mm, p=0.004) and in those with diabetes (0.81mm vs. 0.67mm, p<0.001). AI was higher in the older group (24% vs. 0%, p<0.001), but was not affected by diabetes status. On multivariate regression analysis, low HDL-cholesterol was the only independent predictor of CIMT in the younger group; triglycerides, heart rate (inverse) and height (inverse) were independent predictors of AI in the same group. CONCLUSION: Dyslipidaemia (low HDL-cholesterol or elevated triglycerides) is independently associated with non-invasive measures of cardiovascular disease in a relatively healthy and young subgroup of this high-risk population. We propose that triglycerides and low HDL-cholesterol may represent the most useful commonly measured clinical indicators of cardiovascular risk in young, urban Indigenous Australians.