蛋白激酶A RⅡβ结构及功能的研究进展

蛋白激酶A(PKA)是由1个调节亚基(R亚基)二聚体和两个催化亚基(C亚基)组成的四聚体,全酶无活性。R亚基有RⅠα、RⅠβ、RⅡα和RⅡβ4种亚型,分别具有不同的理化性质。RⅡβ亚基氨基端包含1个二聚化/对接结构域,PKA通过此结构域与腺苷酸激酶锚定蛋白结合锚定于细胞的特定位点。羧基端是两个串联的高度保守的环核苷酸结构域,与PKA全酶解聚以及激活有关。两个RC异二聚体通过RⅡβ亚基中的β4~β5环相锚定。细胞质中存在足够的Mg ATP时将导致RⅡβ亚基自身磷酸化。RⅡβ在特定组织表达,主要表达于内分泌腺、脑和脂肪组织。生物信息学分析表明,RⅡβ与其他R亚基的序列有很大差别,只有大约50%的序列相同,提示RⅡβ可能具有不同的生物学功能。因此,目前对PKA RⅡβ的功能及其作用机制的研究已逐渐成为热点。     

纤维连接蛋白对人结肠癌细胞侵袭力的影响

目的研究纤维连接蛋白（fibronectin，FN）对人结肠癌细胞侵袭力的影响，并探讨其中的信号传导机制。方法以递增浓度的FN刺激结肠癌细胞株Colo320，以免疫沉淀和蛋白质印迹法检测结肠癌细胞内黏着斑激酶（focal adhesion kinase，FAK）第397位酪氨酸（tyr-397）磷酸化的状况．以改良Boyden小室法检测相应的细胞侵袭力变化。设计反义寡核苷酸阻断FAK蛋白质表达，再次观察接受FN刺激后．结肠癌细胞内FAK tyr-397磷酸化状况及细胞侵袭力的改变。结果FN能够促进Colo320 FAK tyr-397磷酸化，在一定范围内具有剂量依赖性。FN浓度达10nmol／L时．此作用最显著，继续增加FN浓度．FAK tyr-397磷酸化不再呈现继续增强趋势．当浓度达到100nmol／L时，磷酸化程度反而有所下降；FN可以增强细胞侵袭力，同样在一定范围内具有剂量依赖性；反义寡核苷酸干预后．结肠癌细胞内FAK tyr-397磷酸化及细胞侵袭力均有显著降低（P〈0.01）。结论FN可以有效增强结肠癌细胞的侵袭力，其作用是通过FN—FAK信号通路实现的，FAK的活性形式是其酪氨酸位点的磷酸化．阻断FAK的表达町以减弱FN促进细胞侵袭的作用。     

Tau pathology modulates Pin1 post-translational modifications and may be relevant as biomarker

A prerequisite to dephosphorylation at Ser–Pro or Thr–Pro motifs is the isomerization of the imidic peptide bond preceding the proline. The peptidyl-prolyl cis/trans isomerase named Pin1 catalyzes this mechanism. Through isomerization, Pin1 regulates the function of a growing number of targets including the microtubule-associated tau protein and is supposed to be deregulated Alzheimer's disease (AD). Using proteomics, we showed that Pin1 is posttranslationally modified on more than 5 residues, comprising phosphorylation, N-acetylation, and oxidation. Although Pin1 expression remained constant, Pin1 posttranslational two-dimensional pattern was modified by tau overexpression in a tau-inducible neuroblastoma cell line, in our THY-Tau22 mouse model of tauopathy as well as in AD. Interestingly, in all of these systems, Pin1 modifications were very similar. In AD brain tissue when compared with control, Pin1 is hyperphosphorylated at serine 16 and found in the most insoluble hyperphosphorylated tau fraction of AD brain tissue. Furthermore, in all tau pathology conditions, acetylation of Pin1 may also contribute to the differences observed. In conclusion, Pin1 displays several posttranslational modifications, which are specific in tauopathies and may be useful as biomarker.     

Ser9 phosphorylation causes cytoplasmic detention of I2/SET in Alzheimer disease

The nuclear protein I₂^PP2A/SET, an endogenous inhibitor of protein phosphatase-2A (PP2A), is increased and translocated to the cytoplasm in the neurons of Alzheimer's disease (AD) brains, and PP2A activity in cytoplasm is compromised. However, it is not fully understood how SET is retained in the cytoplasm. By generating a phosphorylation site-specific antibody, we found in the present study that SET is phosphorylated at Ser9, by which it is accumulated in the cytoplasm of the AD brains. Further studies demonstrate that both the phosphor-mimic and casein kinase (CK)II-mediated phosphorylation at Ser9 interferes with the formation of the SET/importin-α/importin-β complex, and thus inhibits SET nuclear import and induces the cytoplasmic detention of SET. Interestingly, Ser9 is nested in the center of the sequence ⁶AKVSKK¹¹ of SET, which is consistent with a classical nuclear localization signal (NLS). To test whether ⁶AKVSKK¹¹ is a new NLS of SET, we mutated SET lysine 7, lysine 10, and lysine 11 to alanine acid (K7A, K10A, K11A) respectively, and expressed these mutants in HEK293/tau cells. We found that expression of SET (K11A) led to a nuclear import defect of SET, and application of a synthesized peptide Tat-AAKVSKKE that can competitively bind to importin α/β resulted in cytoplasmic detention of SET. Finally, phosphorylation of SET aggravates PP2A inhibition and leads to tau hyperphosphorylation. In conclusion, the current study has identified a novel mechanism that causes cytoplasmic detention of SET with a new NLS-dependent CKII-associated phosphorylation of Ser9, suggesting that inhibition of CKII arrests cytoplasmic accumulation of SET and thus preserves PP2A activity in AD brains.     

Cyclic adenosine monophosphate modulation of contractility via slow Ca2+ channels in chick heart

The catecholamines exert a positive inotropic effect associated with elevated tissue cyclic AMP levels and possibly with increase in the number of membrane slow cationic channels available for voltage activation. In the present study, catecholamines (isoproterenol, dopamine and dobutamine) were tested for their ability to affect the maximum upstroke velocity (+ V_max) of the slow action potentials, the first derivative ($\text{d}\text{T}\text{d}\text{t}$) of developed tension accompanying the slow responses, and the tissue cyclic AMP levels in the ventricular myocardium of isolated perfused chick hearts. To study the slow channels exclusively, the fast Na⁺ channels were voltage inactivated by elevated (25 mm) K⁺. In this condition of functional removal of the fast channels, the heart could not be excited by intense electrical stimulation. It was found that these catecholamines induced slow action potentials accompanied by contractions. Elevation of the concentration of these agents produced increases in + V_max, $\text{d}\text{T}\text{d}\text{t}$, and cyclic AMP in a dose-dependent fashion; a close correlation was obtained between the cyclic AMP level, + V_max and $\text{d}\text{T}\text{d}\text{t}$. These results support the hypothesis that the increases in + V_max of the slow action potentials and in contraction are explained by increase in the number of available slow channels mediated by intracellular cyclic AMP levels, and the resulting increase in the Ca²⁺ influx.     

A mutation in the N-terminus of troponin I that is associated with hypertrophic cardiomyopathy affects the Ca(2+)-sensitivity, phosphorylation kinetics and proteolytic susceptibility of troponin

The first human cardiac troponin I (hcTnI) mutation in the N-terminal 32 residue region, R21C (arginine residue number 21 mutated to cysteine), which has been linked to hypertrophic cardiomyopathy (HCM), has recently been reported. The effect of this mutation on the physiological function of hcTnI was investigated. Human cTnI R21C (in the absence or presence of troponin T and troponin C) was phosphorylated by protein kinase A (PKA) at a significantly slower rate than wild-type hcTnI. In skinned fiber studies, the TnI R21C mutant showed a large increase in Ca(2+)-sensitivity of force development when compared to wild-type TnI (DeltapCa(50)=0.33). Phosphorylation of skinned fibers containing TnI R21C by PKA resulted in a significantly smaller decrease in the Ca(2+)-sensitivity of force development when compared to phosphorylation of fibers containing wild-type TnI. The decreased sensitivity of TnI R21C to PKA is most likely due to a decreased ability of PKA to phosphorylate this TnI rather than conformational problems within this TnI. In addition, skinned fibers were found to contain an endogenous kinase that is capable of phosphorylating wild-type TnI. However, the endogenous kinase activity did not affect the Ca(2+)-sensitivity of force development, the Hill coefficient or maximal force of these skinned fibers. Actomyosin ATPase assays showed that the R21C mutation did not affect the inhibitory properties of TnI or the maximal ATPase activity. TnI R21C was also found to be more susceptible to proteolysis by calpain II than wild-type TnI. These results suggest that this R21C mutation in TnI affects the Ca(2+)-sensitizing effect of Tn, the ability of TnI to be readily phosphorylated by PKA and the stability of TnI to calpain. The results also suggest that the N-terminal region may have important roles such as modulating the Ca(2+)-sensitivity of force-development.     

结直肠癌组织中Thr187磷酸化p27kip1的表达及临床意义

目的 探讨第187位苏氨酸（Thr187）磷酸化的细胞周期素依赖性激酶抑制蛋白（p27kip1,简称p27）（P-p27Thr187）在结直肠癌发生、发展中的作用及其与结直肠癌临床病理特征的关系。方法 收集行手术切除的80例结直肠癌组织标本,以及6对新鲜结直肠癌和相应癌旁正常组织标本;采用免疫组织化学EnVision二步法和蛋白质印迹法检测结直肠癌组织及相应癌旁正常组织中P-p27Thr187和p27的表达;并分析其与结直肠癌临床病理特征的关系。结果免疫组织化学和蛋白质印迹结果均显示,P-p27Thr187在结直肠癌组织中的阳性表达率明显高于癌旁正常组织;结直肠癌组织中P-p27Thr187阳性表达率为100.0%（80/80）,高表达者52例（65.0%,52/80）,低表达者28例（35.0%,28/80）;而癌旁正常组织中P-p27Thr187阳性表达率为13.8%（11/80）,与结直肠癌组织比较差异有统计学意义（P＜0.05）;p27在结直肠癌组织中的阳性表达率为48.8%（39/80）,癌旁正常组织中为90.0%（72/80）,两组比较差异有统计学意义（P＜0.05）;P-p27Thr187阳性表达与肿瘤细胞分化程度和TNM分期有关（均P＜0.05）;P-p27Thr187表达强度与细胞增殖指标Ki-67表达强度呈正相关（r=0.487,P＜0.05）,与p27表达强度呈负相关（r=-0.394,P＜0.05）。结论结直肠癌组织中P-p27Thr187的异常表达可能参与了结直肠癌的发生、发展过程,其有助于综合评估结直肠癌的生物学行为,对结直肠癌的预后判断及诊治具有一定的参考价值。     

p53-Ser~(392)的磷酸化在肿瘤治疗中的作用

磷酸化是p53转录后修饰最常见的方式,但对p53-Ser392的磷酸化在肿瘤治疗中的作用及具体机制知之甚少。我们就p53-Ser392的磷酸化状态对野生型及突变型p53功能的影响、放疗化疗因素及蛋白激酶对p53-Ser392的磷酸化水平的调节和p53-Ser392的磷酸化研究意义进行了阐述。