ABSTRACT
BACKGROUND: Reactive Oxygen species have been known to be a key factor to promote atherosclerosis. DDR2(Discoidin Domain Receptor 2) is a cell surface receptor tyrosine kinase which is activated by fiber collagen. Recently, DDR2 was suggested to be involved in activation of smooth muscle cell in blood vessel of atherosclerosis. METHODS: The effect of antioxidant, N-acetyl cysteine and H2O2(Hydrogen peroxide) in the activation of DDR2 by collagen was studied using HEK293 cells expressing DDR2. The direct activation of DDR2 tyrosine kinase domain by tyrosine phosphorylation upon the treatment of H2O2 was analysed after the kinase domain was expressed in sf9 cells. RESULTS: H2O2 enhanced DDR2 auto-phosphorylation and its cellular signaling to induce MMP-1 expression. However N-acetyl cysteine suppressed the DDR2 activation. The reactive oxygen induced tyrosine phosphorylation in DDR2 tyrosine kinase domain to activate its tyrosine kinase activity. CONCLUSIONS: DDR2 activity can be up-regulated by oxidative stress and this provides a mechanism that DDR2 plays a critical role when reactive oxygen species promote atherosclerosis. Therefore inhibition of the activated DDR2 could be a new therapeutic strategy for atherosclerosis.
Subject(s)
Atherosclerosis , Blood Vessels , Collagen , Cysteine , HEK293 Cells , Myocytes, Smooth Muscle , Oxidative Stress , Oxygen , Phosphorylation , Phosphotransferases , Protein-Tyrosine Kinases , Reactive Oxygen Species , Sf9 Cells , TyrosineABSTRACT
CDK2 and CDK4 known promoter of cell cycling catalyze phosphorylation of RB protein. Enzyme specificity between two CDKs that work at a different cell cycle phase is not clearly understood. In order to define kinase properties of CDK2 and CDK4 in complex with cycline A or cycline D1 in relation to their respective role in cell cycling regulation, we examined enzymatic properties of both CDK4/cycline D1 and CDK2/cycline A in vitro. Association constant, Km for ATP in CDK4/cyclin D1 was found as 418 micrometer, a value unusually high whereas CDK2/cyclin A was 23 micrometer, a value close to most of other regulatory protein kinases. Turnover value for both CDK4/cyclin D1 and CDK2/cyclin A were estimated as 3.4 and 3.9 min(-1)respectively. Kinetic efficiency estimation indicates far over one order magnitude less efficiency for CDK4/cyclin D1 than the value of CDK2/cycline A (9.3 pM(-1)min(-1)and 170 pM(-1)min(-1)respectively). In addition, inhibition of cellular CDK4 caused increase of cellular levels of ATP, even though inhibition of CDK2 did not change it noticeably. These data suggest cellular CDK4/cyclin D1 activity is tightly associated with cellular ATP concentration. Also, analysis of phosphorylated serine/threonine sites on RB catalyzed by CDK4/cyclin D1 and CDK2/cyclin A showed significant differences in their preference of phosphorylation sites in RB C-terminal domain. Since RB is known to regulate various cellular proteins by binding and this binding is controlled by its phosphorylation, these data shown here clearly indicate significant difference in their biochemical properties between CDK4/cyclin D1 and CDK2/cyclin A affecting regulation of cellular RB function.