Phosphorylated p38MAPK specific antibodies cross-react with sarkosyl-insoluble hyperphosphorylated tau proteins

Neurofibrillary tangles (NFT) accumulated in Alzheimer's diseases and related disorders contain hyperphosphorylated tau and display immunoreactivity for active forms of various kinases. To understand the role of p38MAPK (mitogen-activated protein kinase) in NFT formation, we have studied a transgenic (Tg) mouse model of tauopathy, JNPL3, that expresses P301L mutant tau, and bigenic mice, TAPP, generated by cross-breeding of JNPL3 with Tg2576 mice. Age-matched non-Tg mice (NTg), wild-type human tau Tg mice (JN25), and Tg2576 mice were used as controls. Phosphorylated p38MAPK (active form) immunoreactivity was consistently located in NFT and granulovaculolar degeneration in JNPL3 and TAPP mice older than 5 months of age. Unphosphorylated/total-p38MAPK was not detectable in spinal cord and brain sections from 2- to 11-month-old mice, even though JNPL3 mice, but not controls had an age-dependent increase of total-p38MAPK by western blotting. Spinal cord/brain extracts from mice and human with tauopathy were demonstrated to have insignificant amount of active-p38MAPK. However, they contained antiactive-p38MAPK cross-reactive proteins insoluble in sarkosyl and similar to phosphorylated tau in size. Consistently, antiactive-p38MAPK immunoprecipitates displayed tau immunoreactivity, but not total-p38MAPK, and antitau immunoprecipitates displayed active-p38MAPK immunoreactivity. Together, the results indicate that the cross-reactivity of antiactive-p38MAPK antibody with phosphorylated tau is responsible for the immunolabeling of tau-positive inclusion.     

HOG1 MAPK参与调控亚砷酸钠诱导的酵母细胞凋亡

【目的】本研究探讨了HOG1 MAPK在亚砷酸钠诱导酵母细胞凋亡中的作用。【方法】以酵母野生株BY4741及其HOG1突变株(ΔHOG1)为材料,研究了亚砷酸钠对酵母细胞生长、相对存活率和氧化损伤的影响,并采用流式细胞术检测了亚砷酸钠胁迫下酵母细胞凋亡率、ROS水平和线粒体膜电位的变化。【结果】亚砷酸钠可抑制酵母细胞生长,诱导细胞凋亡。在相同处理组中,ΔHOG1对亚砷酸钠更为敏感,表现为细胞存活率降低,凋亡率升高。在亚砷酸钠胁迫过程中,ΔHOG1胞内ROS水平和MDA含量显著高于野生株BY4741,而线粒体膜电位显著低于野生株。【结论】HOG1 MAPK可能通过影响胞内ROS水平和线粒体膜电位的变化调控亚砷酸钠诱导的酵母细胞凋亡。     

cAMP-dependent protein kinase: crystallographic insights into substrate recognition and phosphotransfer.

The crystal structure of ternary and binary substrate complexes of the catalytic subunit of cAMP-dependent protein kinase has been refined at 2.2 and 2.25 A resolution, respectively. The ternary complex contains ADP and a 20-residue substrate peptide, whereas the binary complex contains the phosphorylated substrate peptide. These 2 structures were refined to crystallographic R-factors of 17.5 and 18.1%, respectively. In the ternary complex, the hydroxyl oxygen OG of the serine at the P-site is 2.7 A from the OD1 atom of Asp 166. This is the first crystallographic evidence showing the direct interaction of this invariant carboxylate with a peptide substrate, and supports the predicted role of Asp 166 as a catalytic base and as an agent to position the serine -OH for nucleophilic attack. A comparison of the substrate and inhibitor ternary complexes places the hydroxyl oxygen of the serine 2.7 A from the gamma-phosphate of ATP and supports a direct in-line mechanism for phosphotransfer. In the binary complex, the phosphate on the Ser interacts directly with the epsilon N of Lys 168, another conserved residue. In the ternary complex containing ATP and the inhibitor peptide, Lys 168 interacts electrostatically with the gamma-phosphate of ATP (Zheng J, Knighton DR, Ten Eyck LF, Karlsson R, Xuong NH, Taylor SS, Sowadski JM, 1993, Biochemistry 32:2154-2161). Thus, Lys 168 remains closely associated with the phosphate in both complexes. A comparison of this binary complex structure with the recently solved structure of the ternary complex containing ATP and inhibitor peptide also reveals that the phosphate atom traverses a distance of about 1.5 A following nucleophilic attack by serine and transfer to the peptide. No major conformational changes of active site residues are seen when the substrate and product complexes are compared, although the binary complex with the phosphopeptide reveals localized changes in conformation in the region corresponding to the glycine-rich loop. The high B-factors for this loop support the conclusion that this structural motif is a highly mobile segment of the protein.     

Phospho‐site mapping,genetic and in planta activation studies reveal key aspects of the different phosphorylation mechanisms involved in activation of SnRK2s

Snf1‐related protein kinases 2 (SnRK2s) are major positive regulators of drought stress tolerance. The kinases of this family are activated by hyperosmotic stress, but only some of them are also responsive to abscisic acid (ABA). Moreover, genetic evidence has indicated the ABA‐independence of SnRK2 activation in the fast response to osmotic stress. Although phosphorylation was demonstrated to be crucial for the activation or activity of the kinases of both subgroups, different phosphorylation mechanisms were suggested. Here, using one kinase from each subgroup (SnRK2.6 and SnRK2.10), two phosphorylation sites within the activation loop were identified by mass spectrometry after immunoprecipitation from Arabidopsis cells treated by ABA or osmolarity. By site‐directed mutagenesis, the phosphorylation of only one of the two sites was shown to be necessary for the catalytic activity of the kinase, whereas both sites are necessary for the full activation of the two SnRK2s by hyperosmolarity or ABA. Phosphoprotein staining together with two‐dimensional PAGE followed by immunoblotting indicated distinct phosphorylation mechanisms of the two kinases. While SnRK2.6 seems to be activated through the independent phosphorylation of these two sites, a sequential process occurs in SnRK2.10, where phosphorylation of one serine is required for the phosphorylation of the other. In addition, a subgroup of protein phosphatases 2C which interact and participate in the regulation of SnRK2.6 do not interact with SnRK2.10. Taken together, our data bring evidence for the involvement of distinct phosphorylation mechanisms in the activation of SnRK2.6 and SnRK2.10, which may be conserved between the two subgroups of SnRK2s depending on their ABA‐responsiveness.     

高龄老年心衰患者外周血淋巴细胞GRK2的表达研究

目的:观察高龄老年心衰患者外周血淋巴细胞GRK2的表达及其与心脏射血分数(EF)的关系。方法:选取80岁以上心衰患者16例,按EF分为两组:EF45%组(n=7),EF≥45%组(n=9),80岁以上高龄健康老人作为对照组(n=8),分别抽取外周血2ml,分离淋巴细胞,提取RNA,检测GRK2mRNA的表达。结果:EF45%组的高龄心衰患者外周血淋巴细胞GRK2mRNA的表达高于EF≥45%组(P0.05),且两组明显高于对照组。随着EF的减低,外周血淋巴细胞GRK2mRNA的表达增加。结论:随着EF的减低,高龄心衰患者外周血淋巴细胞GRK2mRNA的表达增加,外周血淋巴细胞GRK2的检测有助于判断高龄老年心衰患者心功能状况及临床治疗心衰疗效的判定。     

Availability of the key metabolic substrates dictates the respiratory response of cancer cells to the mitochondrial uncoupling

Active glycolysis and glutaminolysis provide bioenergetic stability of cancer cells in physiological conditions. Under hypoxia, metabolic and mitochondrial disorders, or pharmacological treatment, a deficit of key metabolic substrates may become life-threatening to cancer cells. We analysed the effects of mitochondrial uncoupling by FCCP on the respiration of cells fed by different combinations of Glc, Gal, Gln and Pyr. In cancer PC12 and HCT116 cells, a large increase in O₂ consumption rate (OCR) upon uncoupling was only seen when Gln was combined with either Glc or Pyr. Inhibition of glutaminolysis with BPTES abolished this effect. Despite the key role of Gln, addition of FCCP inhibited respiration and induced apoptosis in cells supplied with Gln alone or Gal/Gln. For all substrate combinations, amplitude of respiratory responses to FCCP did not correlate with Akt, Erk and AMPK phosphorylation, cellular ATP, and resting OCR, mitochondrial Ca^2 + or membrane potential. However, we propose that proton motive force could modulate respiratory response to FCCP by regulating mitochondrial transport of Gln and Pyr, which decreases upon mitochondrial depolarisation. As a result, an increase in respiration upon uncoupling is abolished in cells, deprived of Gln or Pyr (Glc). Unlike PC12 or HCT116 cells, mouse embryonic fibroblasts were capable of generating pronounced response to FCCP when deprived of Gln, thus exhibiting lower dependence on glutaminolysis. Overall, the differential regulation of the respiratory response to FCCP by metabolic environment suggests that mitochondrial uncoupling has a potential for substrate-specific inhibition of cell function, and can be explored for selective cancer treatment.     

Palbociclib-mediated cell cycle arrest can occur in the absence of the CDK inhibitors p21 and p27

The use of CDK4/6 inhibitors in the treatment of a wide range of cancers is an area of ongoing investigation. Despite their increasing clinical use, there is limited understanding of the determinants of sensitivity and resistance to these drugs. Recent data have cast doubt on how CDK4/6 inhibitors arrest proliferation, provoking renewed interest in the role(s) of CDK4/6 in driving cell proliferation. As the use of CDK4/6 inhibitors in cancer therapies becomes more prominent, an understanding of their effect on the cell cycle becomes more urgent. Here, we investigate the mechanism of action of CDK4/6 inhibitors in promoting cell cycle arrest. Two main models explain how CDK4/6 inhibitors cause G1 cell cycle arrest, which differ in their dependence on the CDK inhibitor proteins p21 and p27. We have used live and fixed single-cell quantitative imaging, with inducible degradation systems, to address the roles of p21 and p27 in the mechanism of action of CDK4/6 inhibitors. We find that CDK4/6 inhibitors can initiate and maintain a cell cycle arrest without p21 or p27. This work clarifies our current understanding of the mechanism of action of CDK4/6 inhibitors and has implications for cancer treatment and patient stratification.     

Molecular basis for activation of G protein-coupled receptor kinases

G protein‐coupled receptor (GPCR) kinases (GRKs) selectively recognize and are allosterically regulated by activated GPCRs, but the molecular basis for this interaction is not understood. Herein, we report crystal structures of GRK6 in which regions known to be critical for receptor phosphorylation have coalesced to stabilize the kinase domain in a closed state and to form a likely receptor docking site. The crux of this docking site is an extended N‐terminal helix that bridges the large and small lobes of the kinase domain and lies adjacent to a basic surface of the protein proposed to bind anionic phospholipids. Mutation of exposed, hydrophobic residues in the N‐terminal helix selectively inhibits receptor, but not peptide phosphorylation, suggesting that these residues interact directly with GPCRs. Our structural and biochemical results thus provide an explanation for how receptor recognition, phospholipid binding, and kinase activation are intimately coupled in GRKs.     

The Rop GTPase: an emerging signaling switch in plants

G proteins are ubiquitous molecular switches in eukaryotic signal transduction, but their roles in plant signal transduction had not been clearly established until recent studies of the plant-specific Rop subfamily of RHO GTPases. Rop participates in signaling to an array of physiological processes including cell polarity establishment, cell growth, morphogenesis, actin dynamics, H₂O₂ generation, hormone responses, and probably many other cellular processes in plants. Evidence suggests that plants have developed unique molecular mechanisms to control this universal molecular switch through novel GTPase-activating proteins and potentially through a predominant class of plant receptor-like serine/threonine kinases. Furthermore, the mechanism by which Rop regulates specific processes may also be distinct from that for other GTPases. These advances have raised the exciting possibility that the elucidation of Rop GTPase signaling may lead to the establishment of a new paradigm for G protein-dependent signal transduction in plants.