丝氨酸/苏氨酸蛋白磷酸酶与DNA损伤应答

DNA损伤应答(DNA damage response,DDR)是维持基因组稳定性的核心机制,对DDR的研究不仅有助于阐明癌症发生发展的机理,同时也为癌症治疗和抗癌新药开发提供生物学基础。蛋白质翻译后修饰,尤其是蛋白激酶介导的磷酸化修饰和蛋白磷酸酶介导的去磷酸化修饰,参与调控绝大多数的生命活动过程,包括DDR。对蛋白激酶ATM/ATR/CHK2/CHK1介导的DDR的研究已经比较透彻,但是对蛋白磷酸酶在DDR中的功能研究还有待加强和深入。比较全面地综述丝氨酸/苏氨酸蛋白磷酸酶在DDR中的功能并探讨在抗癌新药开发中的前景。     

Serine/threonine protein phosphatases: Multi-purpose enzymes in control of defense mechanisms

Depending on the threat to a plant, different pattern recognition receptors, such as receptor-like kinases, identify the stress and trigger action by appropriate defense response development.^1,2 The plant immunity system primary response to these challenges is rapid accumulation of phytohormones, such as ethylene (ET), salicylic acid (SA), and jasmonic acid (JA) and its derivatives. These phytohormones induce further signal transduction and appropriate defenses against biotic threats.^3,4 Phytohormones play crucial roles not only in the initiation of diverse downstream signaling events in plant defense but also in the activation of effective defenses through an essential process called signaling pathway crosstalk, a mechanism involved in transduction signals between two or more distinct, “linear signal transduction pathways simultaneously activated in the same cell.”⁵     

Serine/threonine phosphatases in the nervous system.

The cloning and sequence determination of cDNAs encoding different types of serine/threonine protein phosphatases has provided a molecular basis for the protein phosphatase classification proposed by Ingebritsen and Cohen. Each of the phosphatases, phosphatase-1, -2A, -2B and -2C, exists as multiple isozymes raising the possibility that isozymes selectively expressed in different tissues may perform specific functions. The recent discovery of potent toxin inhibitors specific for protein phosphatase-1 and -2A will undoubtedly play an important role in the elucidation of the role of these enzymes in neuronal function.     

Serine/threonine protein phosphatases type 2A and their roles in stress signaling

Serine/threonine protein phosphatases are ubiquitous enzymes in all eukaryotes but many of their physiological roles in plants remain unknown. The available results have demonstrated critical functions for these enzymes in the regulation of adaptive stress responses, and recent studies have directed attention to the functional roles of Ser/Thr phosphatases type 2A (PP2A) as components of stress signaling pathways. This review is focused primarily on plant PP2As and their participation in the control of biotic and abiotic stress responses.Key words: protein phosphatases type 2A, PP2A, biotic stress, abiotic stress, signaling, okadaic acid     

Serine/threonine protein kinases and apoptosis

Over the past decade, our understanding of apoptosis, or programmed cell death, has increased greatly, with the identification of some of the major components of the apoptotic programme and the processes regulating their activation. Although apoptosis is an intrinsic process present in all cells, it can be regulated by extrinsic factors, including hormones, growth factors, cell surface receptors, and cellular stress. The actions of both pro- and antiapoptotic factors are often affected by modulation of the phosphorylation status of key elements of the apoptotic process. This minireview will focus on the role of protein kinases in apoptosis. Apoptosis is a multistep process and protein kinases have been implicated both in the upstream induction phase of apoptosis and in the downstream execution stage, as the direct targets for caspases. Due to the space constraints of this review it is not possible to discuss all of the kinases involved in the apoptotic process and we have focused here on the role of the serine/threonine protein kinases. The kinases of this family that have been suggested to play a role in apoptosis are the mitogen-activated protein kinase (MAPK) family, specifically p42/44 ERK, p38 MAPK and c-Jun N-terminal kinase (JNK), cyclic AMP-dependent protein kinase (PKA), protein kinase B (PKB), or Akt and protein kinase C (PKC). We have also considered briefly the potential for the regulation of these kinases by tyrosine protein kinases, such as c-abl.     

Serine/threonine protein phosphatases PP1 and PP2A are key players in apoptosis

The reversible phosphorylation of proteins controlled by protein kinases and protein phosphatases is a major mechanism that regulates a wide variety of cellular processes. In contrast to C. elegans, recent studies in mammalian cells have highlighted a major role of serine/threonine protein phosphorylation in apoptosis. To illustrate the importance of dephosphorylation processes in apoptosis, this review will focus on recent studies suggesting that the interaction of the serine/threonine protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) with certain regulators of the Bcl-2 family is critically involved in the control of apoptosis.     

Serine/threonine protein kinases in Drosophila

The study of serine/threonine kinases in Drosophila is coming of age. Recently several kinases have been identified and their role in cell determination has been established. This review discusses these recent findings and describes the potential for genetic analyses of kinase activity and signal transduction.     

Serine/threonine protein phosphatases in Dictyostelium discoideum: no evidence for type I activity.

Extracts from Dictyostelium discoideum contain type 2A and 2C serine/threonine-specific protein phosphatases with properties very similar to those from mammals according to their sensitivity to okadaic acid and to their dependence for divalent cations. In contrast, no type 1 protein phosphatase is found at any time of development, neither in the cytosolic nor in the particulate fraction, using glycogen phosphorylase a, casein, histone or the non-proteinous 4-Methylumbelliferyl phosphate as substrates. Both type 2A and 2C protein phosphatase activities remain constant throughout the development cycle.     

Comparison of K-Cl cotransport expression in high and low K dog erythrocytes.

K-Cl cotransport plays a crucial role in regulatory volume decrease of erythrocytes. K-Cl cotransport activities in dog erythrocytes with an inherited high Na-K pump activity (HK) and normal erythrocytes (LK) were compared. Nitrite (NO(2)) stimulated K-Cl cotransport activity in HK cells around 14-fold at 2.4 mM, and it also increased the Km value of this cotransporter. Real-time PCR and western blot analysis revealed that K-Cl cotransporter 1 was dominant, and that the quantity of K-Cl cotransporter 1 protein was comparable between HK and LK erythrocytes. These results suggest that the difference in cotransport activity was not caused by the amount of K-Cl cotransport protein but by a difference in the regulation system, which is susceptible to oxidant.     

K-Cl cotransport in vascular smooth muscle and erythrocytes: possible implication in vasodilation

K-Cl cotransport, theelectroneutral-coupled movement of K and Cl ions, plays an importantrole in regulatory volume decrease. We recently reported that nitrite,a nitric oxide derivative possessing potent vasodilation properties,stimulates K-Cl cotransport in low-K sheep red blood cells (LK SRBCs).We hypothesized that activation of vascular smooth muscle (VSM) K-Clcotransport by vasodilators decreases VSM tension. Here we tested thishypothesis by comparing the effects of commonly used vasodilators,hydralazine (HYZ), sodium nitroprusside, isosorbide mononitrate, andpentaerythritol, on K-Cl cotransport in LK SRBCs and in primarycultures of rat VSM cells (VSMCs) and of HYZ-induced K-Clcotransport activation on relaxation of isolated porcine coronaryrings. K-Cl cotransport was measured as the Cl-dependent K efflux or Rbinflux in the presence and absence of inhibitors for other K/Rbtransport pathways. All vasodilators activated K-Cl cotransport in LKSRBCs and HYZ in VSMCs, and this activation was inhibited by calyculinand genistein, two inhibitors of K-Cl cotransport. KT-5823, a specificinhibitor of protein kinase G, abolished the sodiumnitroprusside-stimulated K-Cl cotransport in LK SRBCs, suggestinginvolvement of the cGMP pathway in K-Cl cotransport activation.Hydralazine, in a dose-dependent manner, and sodium nitroprussiderelaxed (independently of the endothelium) precontractedarteries when only K-Cl cotransport was operating and other pathwaysfor K/Rb transport, including the Ca-activated K channel, wereinhibited. Our findings suggest that K-Cl cotransport may be involvedin vasodilation.

     

Brain protein serine/threonine phosphatases.

All of the known protein serine/threonine phosphatases are expressed in the brain. These enzymes participate in a variety of signaling pathways that modulate neuronal activity. The multifunctional activity of many serine/threonine phosphatases is achieved through their association with targeting proteins. Identification and analysis of targeting molecules has led to new insights into the functions of protein phosphatases in neuronal signaling. The recent use of transgenic mice has also increased our understanding of the physiological roles of these enzymes in the brain.     

Swelling activation of K-Cl cotransport in LK sheep erythrocytes: a three-state process

K-Cl cotransport in LK sheep erythrocytes is activated by osmotic swelling and inhibited by shrinkage. The mechanism by which changes in cell volume are transduced into changes in transport was investigated by measuring time courses of changes in transport after osmotic challenges in cells with normal and reduced Mg concentrations. When cells of normal volume and normal Mg are swollen, there is a delay of 10 min or more before the final steady-state flux is achieved, as there is for swelling activation of K-Cl cotransport in erythrocytes of other species. The delay was shown to be independent of the extent of swelling. There was also a delay after shrinkage inactivation of cotransport. Reducing cellular Mg concentration activates cotransport. Swelling of low-Mg cells activates cotransport further, but with no measurable delay. In contrast, there is a delay in shrinkage inactivation of cotransport in low-Mg cells. The results are interpreted in terms of a three-state model: [formula see text] in which A state, B state, and C state transporters have relatively slow, intermediate, and fast transport rates, respectively. Most transporters in shrunken cells with normal Mg are in the A state. Swelling converts transporters to the B state in the rate-limiting process, followed by rapid conversion to the C state. Reducing cell Mg also promotes the A-- >B conversion. Swelling of low-Mg cells activates transport rapidly because of the initial predominance of B state transporters. The results support the following conclusions about the rate constants of the three-state model: k21 is the rate constant for a Mg-promoted process that is inhibited by swelling; k12 is not volume sensitive. Both k23 and k32 are increased by swelling and reduced by shrinkage; they are rate constants for a single process, whereas k12 and k21 are rate constants for separate processes. Finally, the A-->B conversion entails an increase in Jmax of the transporters, and the B-->C conversion entails an increase in the affinity of the transporters for K.     

K-Cl cotransport in LK sheep erythrocytes: Kinetics of stimulation by cell swelling

Summary The effects of osmotic cell swelling were studied on the kinetics of Cl-dependent K⁺ influx, K–Cl cotransport, in erythrocytes from sheep of the low K⁺ (LK) phenotype. Swelling 25% stimulated transport by increasing maximum velocity (J _max) 1.5-fold and by increasing apparent affinity for external K (K _o ) nearly twofold. Dithiothreitol (DTT) was shown to be a partial, reversible inhibitor of K–Cl cotransport. It inhibited in cells of normal volume by reducingJ _max more than twofold: apparent affinity for K _o was increased by DTT, suggesting that DTT stabilizes the transporter-K _o complex. Cell swelling reduced the extent of inhibition by DTT:J _max was inhibited by only about one-third in swollen cells, and apparent affinity was only slightly affected. This result suggested that DTT does not act directly on the transporter, but on a hypothetical regulator, an endogenous inhibitor. Swelling relieves inhibition by the regulator, and reduces the effect of DTT. Reducing intracellular Mg²⁺, Mg _o , stimulated cotransport. Swelling of low-Mg²⁺ cells stimulated transport further, but only by raising apparent affinity for K _o nearly threefold:J _max was unaffected. Thus effects of swelling onJ _max and apparent affinity are separable processes. The inhibitory effects of Mg _o and DTT were shown to be additive, indicating separate modes of action. There appear to be two endogenous inhibitors: the hypothetical regulator, which holds affinity for K _o , low; and Mg _o , which affectsJ _max perhaps by holding some transporters in an inactive form. Swelling stimulates transport by relieving both types of inhibition.     

Coordinated regulation of Na/H exchange and [K-Cl] cotransport in dog red cells

Swelling-activated [K-Cl] cotransport and shrinkage-activated Na/H exchange were studied in dog red cells with altered internal Mg or Li content. The two pathways responded in a coordinated fashion. When cells were depleted of Mg, [K-Cl] cotransport was stimulated and Na/H exchange was inhibited. Raising internal Mg had the opposite effect: [K-Cl] cotransport was inhibited and Na/H exchange was stimulated. Li loading, previously shown to stimulate Na/H exchange, inhibited [K-Cl] cotransport. From these reciprocal effects and from other evidence, we surmise that the regulation of Na/H exchange and [K-Cl] cotransport is conducted and coordinated by a discrete mechanism that responds to changes in cell volume and is sensitive to cytoplasmic Mg and Li concentrations.     

Regulation of K-Cl cotransport during reticulocyte maturation and erythrocyte aging in normal and sickle erythrocytes

The age/density-dependent decrease in K-Cl cotransport (KCC), PP1 and PP2A activities in normal and sickle human erythrocytes, and the effect of urea, a known KCC activator, were studied using discontinuous, isotonic gradients. In normal erythrocytes, the densest fraction (d 33.4 g/dl) has only about 5% of the KCC and 4% of the membrane (mb)-PP1 activities of the least-dense fraction (d 24.7 g/dl). In sickle and normal erythrocytes, density-dependent decreases for mb-PP1 activity were similar (d_50% 28.1 ± 0.4 vs. 27.2 ± 0.2 g/dl, respectively), whereas those for KCC activity were not (d_50% 31.4 ± 0.9 vs. 26.8 ± 0.3 g/dl, respectively, P = 0.004). Excluding the 10% least-dense cells, a very tight correlation exists between KCC and mb-PP1 activities in normal (r² = 0.995) and sickle erythrocytes (r² = 0.93), but at comparable mb-PP1 activities, KCC activity is higher in sickle erythrocytes, suggesting a defective, mb-PP1-independent KCC regulation. In normal, least-dense but not in densest cells, urea stimulates KCC (two- to fourfold) and moderately increases mb-PP1 (20–40%). Thus mb-PP1 appears to mediate part of urea-stimulated KCC activity. phosphorylation; protein phosphatase; urea; cell size; density