Identification and functional analysis of two Ca2+-binding EF-hand motifs in the B"/PR72 subunit of protein phosphatase 2A

Protein phosphatase 2A (PP2A) is a multifunctional serine/threonine phosphatase that is critical to many cellular processes including cell cycle regulation and signal transduction. PP2A is a heterotrimer containing a structural (A) and catalytic (C) subunit, associated with one variable regulatory or targeting B-type subunit, of which three families have been described to date (B/PR55, B'/PR61, and B"/PR72). We identified two functional and highly conserved Ca(2+)-binding EF-hand motifs in human B"/PR72 (denoted EF1 and EF2), demonstrating for the first time the ability of Ca(2+) to interact directly with and regulate PP2A. EF1 and EF2 apparently bind Ca(2+) with different affinities. Ca(2+) induces a significant conformational change, which is dependent on the integrity of the motifs. We have further evaluated the effects of Ca(2+) on subunit composition, subcellular targeting, catalytic activity, and function during the cell cycle of a PR72-containing PP2A trimer (PP2A(T72)) by site-directed mutagenesis of either or both motifs. The results suggest that integrity of EF2 is required for A/PR65 subunit interaction and proper nuclear targeting of PR72, whereas EF1 might mediate the effects of Ca(2+) on PP2A(T72) activity in vitro and is at least partially required for the ability of PR72 to alter cell cycle progression upon forced expression.     

Baculovirus expression, purification, and characterization of human protein phosphatase 2A catalytic subunits alpha and beta

Protein phosphatase 2A (PP2A) contains a 36-kDa catalytic subunit (PP2Ac), a 65-kDa structural subunit (PR65/A), and a regulatory B subunit. The core enzyme consists of the structural and catalytic subunits. The catalytic subunit exists as two closely related isoforms, alpha and beta. Several natural toxins, including okadaic acid (OA) and microcystins, specifically inhibit PP2A. To obtain biologically active recombinant PP2A and to compare the properties of the PP2A catalytic subunit alpha and beta isoforms, we expressed human PP2Acalpha and cbeta in High Five insect cells. The recombinant PP2Acalpha and cbeta possess similar phosphatase activities using p-NPP and phosphopeptide as substrates and are strongly inhibited by OA and microcystin-LR to similar degrees. In addition, PP2Acalpha or cbeta was co-expressed with PR65/A and co-purified as a core dimer, PP2AD (Aalpha/calpha and Aalpha/cbeta) with PR65alpha/Aalpha. The recombinant PP2AD bound to the B subunit in vitro. These results show that the recombinant PP2Acalpha and cbeta are identical in their ability to associate with the A and B subunits, in their phosphatase activities, and in carboxyl-methylation. Furthermore, our results show that High Five insect cells can produce biologically active recombinant PP2A, which should be a valuable tool for detecting natural toxins and investigating the mechanism of PP2A catalysis and other protein interactions.     

Structure of the Ca2+-dependent PP2A heterotrimer and insights into Cdc6 dephosphorylation

The B″/PR72 family of protein phosphatase 2A (PP2A) is an important PP2A family involved in diverse cellular processes, and uniquely regulated by calcium binding to the regulatory subunit. The PR70 subunit in this family interacts with cell division control 6 (Cdc6), a cell cycle regulator important for control of DNA replication. Here, we report crystal structures of the isolated PR72 and the trimeric PR70 holoenzyme at a resolution of 2.1 and 2.4 Å, respectively, and in vitro characterization of Cdc6 dephosphorylation. The holoenzyme structure reveals that one of the PR70 calcium-binding motifs directly contacts the scaffold subunit, resulting in the most compact scaffold subunit conformation among all PP2A holoenzymes. PR70 also binds distinctively to the catalytic subunit near the active site, which is required for PR70 to enhance phosphatase activity toward Cdc6. Our studies provide a structural basis for unique regulation of B″/PR72 holoenzymes by calcium ions, and suggest the mechanisms for precise control of substrate specificity among PP2A holoenzymes.     

The dependence receptor UNC5H2/B triggers apoptosis via PP2A-mediated dephosphorylation of DAP kinase

The UNC5H dependence receptors promote apoptosis in the absence of their ligand, netrin-1, and this is important for neuronal and vascular development and for limitation of cancer progression. UNC5H2 (also called UNC5B) triggers cell death through the activation of the serine-threonine protein kinase DAPk. While performing a siRNA screen to identify genes implicated in UNC5H-induced apoptosis, we identified the structural subunit PR65β of the holoenzyme protein phosphatase 2A (PP2A). We show that UNC5H2/B recruits a protein complex that includes PR65β and DAPk and retains PP2A activity. PP2A activity is required for UNC5H2/B-induced apoptosis, since it activates DAPk by triggering its dephosphorylation. Moreover, netrin-1 binding to UNC5H2/B prevents this effect through interaction of the PP2A inhibitor CIP2A to UNC5H2/B. Thus we show here that, in the absence of netrin-1, recruitment of PP2A to UNC5H2/B allows the activation of DAPk via a PP2A-mediated dephosphorylation and that this mechanism is involved in angiogenesis regulation.     

Protein phosphatase 2A positively regulates Ras signaling by dephosphorylating KSR1 and Raf-1 on critical 14-3-3 binding sites

BACKGROUND: Kinase Suppressor of Ras (KSR) is a conserved component of the Ras pathway that acts as a molecular scaffold to facilitate signal transmission through the MAPK cascade. Although recruitment of KSR1 from the cytosol to the plasma membrane is required for its scaffolding function, the precise mechanism(s) regulating the translocation of KSR1 have not been fully elucidated. RESULTS: Using mass spectrometry to analyze the KSR1-scaffolding complex, we identify the serine/threonine protein phosphatase PP2A as a KSR1-associated protein and show that PP2A is a critical regulator of KSR1 activity. We find that the enzymatic core subunits of PP2A (PR65A and catalytic C) constitutively associate with the N-terminal domain of KSR1, whereas binding of the regulatory PR55B subunit is induced by growth factor treatment. Specific inhibition of PP2A activity prevents the growth factor-induced dephosphorylation event involved in the membrane recruitment of KSR1 and blocks the activation of KSR1-associated MEK and ERK. Moreover, we find that PP2A activity is required for activation of the Raf-1 kinase and that both Raf and KSR1 must be dephosphorylated by PP2A on critical regulatory 14-3-3 binding sites for KSR1 to promote MAPK pathway activation. CONCLUSIONS: These findings identify KSR1 as novel substrate of PP2A and demonstrate the inducible dephosphorylation of KSR1 in response to Ras pathway activation. Further, these results elucidate a common regulatory mechanism for KSR1 and Raf-1 whereby their localization and activity are modulated by the PP2A-mediated dephosphorylation of critical 14-3-3 binding sites.     

Overlapping binding sites in protein phosphatase 2A for association with regulatory A and alpha-4 (mTap42) subunits

Diverse functions of protein Ser/Thr phosphatases depend on the distribution of the catalytic subunits among multiple regulatory subunits. In cells protein phosphatase 2A catalytic subunit (PP2Ac) mostly binds to a scaffold subunit (A subunit or PR65); however, PP2Ac alternatively binds to alpha-4, a subunit related to yeast Tap42 protein, which also associates with phosphatases PP4 or PP6. We mapped alpha-4 binding to PP2Ac to the helical domain, residues 19-165. We mutated selected residues and transiently expressed epitope-tagged PP2Ac to assay for association with A and alpha-4 subunits by co-precipitation. The disabling H118N mutation at the active site or the presence of the active site inhibitor microcystin-LR did not interfere with binding of PP2Ac to either the A subunit or alpha-4, showing that these are allosteric regulators. Positively charged side chains Lys(41), Arg(49), and Lys(74) on the back surface of PP2Ac are unique to PP2Ac, compared with phosphatases PP4, PP6, and PP1. Substitution of one, two, or three of these residues with Ala produced a progressive loss of binding to the A subunit, with a corresponding increase in binding to alpha-4. Conversely, mutation of Glu(42) in PP2Ac essentially eliminated PP2Ac binding to alpha-4, with an increase in binding to the A subunit. Reciprocal changes in binding because of mutations indicate competitive distribution of PP2Ac between these regulatory subunits and demonstrate that the mutated catalytic subunits retained a native conformation. Furthermore, neither the Lys(41)-Arg(49)-Lys(74) nor Glu(42) mutations affected the phosphatase-specific activity or binding to microcystin-agarose. Binding of PP2Ac to microcystin and to alpha-4 increased with temperature, consistent with an activation energy barrier for these interactions. Our results reveal that the A subunit and alpha-4 (mTap42) require charged residues in separate but overlapping surface regions to associate with the back side of PP2Ac and modulate phosphatase activity.     

Human cytomegalovirus carries serine/threonine protein phosphatases PP1 and a host-cell derived PP2A.

Human cytomegalovirus (CMV), a herpesvirus, is an important cause of morbidity and mortality in immunocompromised patients. When studying hyper-immediate-early events after contact between CMV virions and the cell membrane, we observed a hypophosphorylation of cellular proteins within 10 min. This can be explained in part by our finding that purified CMV contains serine/threonine protein phosphatase activities. Biochemical analyses indicate that this protein phosphatase activity has all characteristics of type 1 and 2A protein phosphatases (PP1 and PP2A). Specifically, PP1 accounts for approximately 30% and PP2A accounts for the remaining 70% of the phosphorylase phosphatase activity found. CMV produced in astrocytoma cells stably expressing an amino-terminally tagged PP2A catalytic subunit contained tagged enzyme, thus demonstrating the cellular origin of CMV-associated PP2A. PP2A is specifically found inside the virus, associated with the nucleocapsid fraction. Western blot (immunoblot) analysis of purified virus revealed the presence of the catalytic subunits of PP2A and PP1. Furthermore, the catalytic subunit of PP2A appears to be complexed to the regulatory subunits PR65 and PR55, which is also the most abundant configuration of this enzyme found in the host cells. Incubation of virus with okadaic acid before contact of CMV with cells prevented hypophosphorylation of cellular proteins, thus demonstrating the role of CMV-associated phosphatases in this phenomenon. CMV can thus transport an active enzyme from one cell to another.     

Activation of Fas inhibits heat-induced activation of HSF1 and up-regulation of hsp70.

Activation of heat shock factor (HSF) 1-DNA binding and inducible heat shock protein (hsp) 70 (also called hsp72) expression enables cells to resist various forms of stress and survive. Fas, a membrane-bound protein, is a central proapoptotic factor; its activation leads to a cascade of events, resulting in programmed cell death. These two mechanisms with contradictory functions, promoting either cell survival or death, were examined for their potential to inhibit each other's activation. Induction of FAS-mediated signaling was followed by a rapid decrease in HSF1-DNA binding and inducible hsp70 expression. Inhibition of HSF1-DNA binding was demonstrated to be based on absent hyperphosphorylation of HSF1 during FAS signaling. These effects of FAS activation on the HSF1/hsp70 stress response were blocked by ICE (caspase 1) inhibitors, suggesting an ICE-mediated process. Furthermore, inhibition of HSF1/hsp70 was accompanied by an increase in apoptosis rates from 20% to 50% in response to heat stress. When analyzing the effects of HSF1/hsp70 activation on Fas-mediated apoptosis, protection from apoptosis was seen in cells with induced hsp70 protein levels, but not in cells that were just induced for HSF1-DNA binding. Thus, we conclude that inhibition of HSF1/hsp70 stress response during Fas-mediated apoptosis and vice versa may facilitate a cell to pass a previously chosen pathway, stress resistance or apoptosis, without the influence of inhibitory signals.     

大鼠再生肝中hsbp1、hsf1、hsf2、hsp70表达水平改变的分析

在克隆了大鼠热休克因子结合蛋白1基因(hsbp1)全长cDNA基础上,进一步分析它在肝再生中作用。用SD纯系大鼠为材料,按Higgens等方法建立大鼠部分肝切除(PH)模型;用原位杂交等方法分析hsbp1在肝再生中表达变化;用基因表达谱芯片分析hsbp1、hsf1、hsf2和hsp70在肝再生中表达变化。原位杂交和基因表达谱芯片分析表明,PH后6h和66-144h,hsbp1表达发生了有意义上调;8-16h,hsf1表达发生了有意义上调;2-16h,hsf2表达发生了有意义上调;0.5-24h,hsp70表达发生了有意义上调。假手术(只打开腹腔和翻动肝叶,但不进行部分肝切除)后0.5-2h,hsbp1表达发生了有意义下调;8-16h,hsf1表达发生了有意义上调;0-144h,hsf2未发生有意义表达变化;0.5-30h,hsp70表达发生了有意义上调。根据实验结果推测,PH后hsbp1表达上调可增加细胞内HSBP1量,促进生长、发育、分化相关基因表达和再生肝的组织结构功能重建;(假)手术后hsbp1表达下调可减少细胞内HSBP1量,有利于HSF1上调hsp70表达,提高机体和肝脏抗损伤能力。     

大鼠再生肝中hsbp1、hsf1、hsf2、shp70表达水平改变的分析

在克隆了大鼠热休克因子结合蛋白1基因（hsbp1）全长cDNA基础上,进一步分析它在肝再生中作用。用SD纯系大鼠为材料。按Higgens等方法建立大鼠部分肝切除（PH）模型;用原位杂交等方法分析凤6pJ在肝再生中表达变化;用基因表达谱芯片分析凤如1、hsf1、如,2和hsp70在肝再生中表达变化。原位杂交和基因表达谱芯片分析表明。PH后6h和66-144h,hsbp1表达发生了有意义上调;8-16h,nsf1表达发生了有意义上调;2-16h,hsf2表达发生了有意义上调;0．5—24h,hsp70表达发生了有意义上调。假手术（只打开腹腔和翻动肝叶。但不进行部分肝切除）后0．5-2h,hsbp1表达发生了有意义下调;8—16h,hsf1表达发生了有意义上调;0—144h,hsf2未发生有意义表达变化;0．5—30h,hsp70表达发生了有意义上调。根据实验结果推测,PH后hsbp1表达上调可增加细胞内HSBP1量。促进生长、发育、分化相关基因表达和再生肝的组织结构功能重建;（假）手术后hsbp1表达下调可减少细胞内HSBP1量,有利于HSF1上调hsp70表达,提高机体和肝脏抗损伤能力。