G Protein-coupled Receptor Kinase 5 Of The Regulation Of Apoptosis And Its Mechanism

G protein-coupled receptors (GPCRs), the largest family of membrane receptors, transduce a large number of extracellular signals including hormones, neurotransmitters, odors and drugs to the interior of cells. G-protein-coupled receptor kinases (GRKs) comprise a family of serine/threonine protein kinases that bind and phosphorylate cytoplasmic domain of GPCRs upon agonist challenge, thus inhibit the persisting activation of receptors, mediate desensitization of GPCR signaling, and play an important role in the following internalization and signaling of GPCRs. Seven isoforms of GRKs (GRK1-7) have been identified, divided into three subfamilies. Being the best-characterized member of the GRK4 subfamily, GRK5 is found most abundantly in heart and lung, while expressed wildly in brain, musle and peripheral tissues. To this day, a variety of GPCRs have been found to be regulated by GRK5 selectively. Besides their well-known role as negetive regulator of GPCRs, there might be additional physiological significance of GRKs. GRK5 has been also reported to interact with and regulate nonreceptor substrates, have a functional nuclear localization sequence (NLS), capable of processing nuclear-cytoplasmic shuttling and regulate transcriptional factors. Furthermore, recent evidence indicates that members of GRK family may also serve as potential contributors of physiological processes, such as apoptosis and cell cycle control. However, detailed knowledge of the molecular mechanisms of these regulation remains to be investigated.The tumor suppressor p53 primarily functions as a transcriptional factor and mediates a variety of downstream functions, including cell-cycle arrest or apoptotic cell death in response to DNA damage, cell growth, differentiation, tumorigenesis and senescence. The regulation of p53 level is important to its function. In non-stressed cells, p53 is kept at low levels by ubiquitination and following proteolysis. Phosphorylation of p53 is crucial for the regulation of p53 stabilization and activation. Our current study investigated the effect of GRK5 on apoptotic death in response to DNA damage, and revealed a novel function for GRK5 in the regulation of p53 through its kinase activity. Our results showed that:1. GRK5 interacts with and phosphorylate p53 at Thr55, resulting in p53 degradation. We demonstrated that GRK5 can interact and phosphorylate p53 at Thr55 in vitro and in vivo. Substitution of Thr55 with an alanine residue (T55A), or using heparin, the inhibitor of GRK family, abolish the phosphorylation notablely. Treatment with GRK5 siRNA lead to decrease in the phosphorylation of endogenous p53 at Thr55, and increase in the protein level of p53, while overexpression of GRK 5 lead to the opposite effect. The p53 mRNA level was not altered by GRK5 siRNA. when the Mdm2-ubiquitin-proteasome degradation system was blocked, the effect of GRK5 siRNA on p53 level was eliminated, indicating that GRK5 regulates p53 level via Mdm2-mediated degradation. We also found that overexpression of GRK5, but not GRK5 kinase dead mutant, increased ubiquitination levels of p53, which can be abolished by substitution of Thr55 with an alanine. Furthermore, the degradation rate of p53 was increased in cells overexpressing GRK5, while treatment with GRK5 siRNA lead to the opposite effect. These results suggesting that GRK5 promotes ubiquitination and proteolysis of p53 via phosphorylation.2. GRK5 regulated p53-dependent apoptosis and G1 arrest. We found that treatment of GRK5 siRNA increased the incidence of tumor cell apoptosis in response to DNA damage. Overexpression of GRK5 inhibite apoptosis in p53+/+cells, but not in p53-/-cells, indicating GRK5 regulates apoptosis mainly through p53. Overexpression of GRK5 kinase dead mutant had no such effect, suggesting the regulation of apoptosis was kinase activity-dependent. We also analysed the role of GRK5 on cell cycle and proliferation. Treatment of GRK5 siRNA resulted in G1 phase arrest and inhibition of cellproliferation. On the contrary, overexpression of GRK5 promoted cell proliferation, which was dependent on p53 phosphorylation. These data suggested that GRK5 regulates apoptosis and cell cycle, at least in part, through p53 phosphorylation.3. Increased expression of p53 and p53 target gene Bax was found in multiple organs of GRK5 knock out mice.In addition, whole-body y-irradiation resulted in a higher apoptosis percentage in thymocytes of GRK5 knockout mice, which support an essential role for GRK5 as a negative regulator of p53 under physiological conditions.Thus we describe a previously unknown function of GRK5, a subunit of GRK family, as a regulator of tumor suprossor p53.We found that GRK5 could regulate p53-mediated apoptosis in response to DNA damage. GRK5 interacts with and phosphorylate p53 at Thr55 in vitro and in vivo. The phosphorylation by GRK5 results in p53 degradation and affects cell G1 arrest and proliferation. Moreover, in mutiple tissue types of the GRK5 knock out mice, we found an increased level of p53. IR induced apoptosis was also enhanced in thymus cells of GRK5 knock out mice. Thus our results uncovered a novel function for GRK5 as a inhibitor of p53 and elucidate its function as a regulator of apoptosis and cell cycle.