Novel Binding of the Mitotic Regulator TPX2 (Target Protein for Xenopus Kinesin-like Protein 2) to Importin-��

Several aspects of mitotic spindle assembly are orchestrated by the Ran GTPase through its modulation of the interaction between spindle assembly factors and importin-α. One such factor is TPX2 that promotes microtubule assembly in the vicinity of chromosomes. TPX2 is inhibited when bound to importin-α, which occurs when the latter is bound to importin-β. The importin-α:β interaction is disrupted by the high RanGTP concentration near the chromosomes, releasing TPX2. In more distal regions, where Ran is predominantly GDP-bound, TPX2 remains bound to importin-α and so is inhibited. Here we use a combination of structural and biochemical methods to define the basis for TPX2 binding to importin-α. A 2.2 Å resolution crystal structure shows that the primary nuclear localization signal (²⁸⁴KRKH²⁸⁷) of TPX2, which has been shown to be crucial for inhibition, binds to the minor NLS-binding site on importin-α. This atypical interaction pattern was confirmed using complementary binding studies that employed importin-α variants in which binding to either the major or minor NLS-binding site was impaired, together with competition assays using the SV40 monopartite NLS that binds primarily to the major site. The different way in which TPX2 binds to importin-α could account for much of the selectivity necessary during mitosis because this would reduce the competition for binding to importin-α from other NLS-containing proteins.     

DC2 and Keratinocyte-associated Protein 2 (KCP2), Subunits of the Oligosaccharyltransferase Complex,Are Regulators of the γ-Secretase-directed Processing of Amyloid Precursor Protein (APP)

The oligosaccharyltransferase complex catalyzes the transfer of oligosaccharide from a dolichol pyrophosphate donor en bloc onto a free asparagine residue of a newly synthesized nascent chain during the translocation in the endoplasmic reticulum lumen. The role of the less known oligosaccharyltransferase (OST) subunits, DC2 and KCP2, recently identified still remains to be determined. Here, we have studied DC2 and KCP2, and we have established that DC2 and KCP2 are substrate-specific, affecting amyloid precursor protein (APP), indicating that they are not core components required for N-glycosylation and OST activity per se. We show for the first time that DC2 and KCP2 depletion affects APP processing, leading to an accumulation of C-terminal fragments, both C99 and C83, and a reduction in full-length mature APP. This reduction in mature APP levels was not due to a block in secretion because the levels of sAPPα secreted into the media were unaffected. We discover that DC2 and KCP2 depletion affects only the γ-secretase complex, resulting in a reduction of the PS1 active fragment blocking Aβ production. Conversely, we show that the overexpression of DC2 and KCP2 causes an increase in the active γ-secretase complex, particularly the N-terminal fragment of PS1 that is generated by endoproteolysis, leading to a stimulation of Aβ production upon overexpression of DC2 and KCP2. Our findings reveal that components of the OST complex for the first time can interact with the γ-secretase and affect the APP processing pathway.     

Metabolic Control of Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII)-mediated Caspase-2 Suppression by the B55β/Protein Phosphatase 2A (PP2A)

High levels of metabolic activity confer resistance to apoptosis. Caspase-2, an apoptotic initiator, can be suppressed by high levels of nutrient flux through the pentose phosphate pathway. This metabolic control is exerted via inhibitory phosphorylation of the caspase-2 prodomain by activated Ca²⁺/calmodulin-dependent protein kinase II (CaMKII). We show here that this activation of CaMKII depends, in part, on dephosphorylation of CaMKII at novel sites (Thr³⁹³/Ser³⁹⁵) and that this is mediated by metabolic activation of protein phosphatase 2A in complex with the B55β targeting subunit. This represents a novel locus of CaMKII control and also provides a mechanism contributing to metabolic control of apoptosis. These findings may have implications for metabolic control of the many CaMKII-controlled and protein phosphatase 2A-regulated physiological processes, because both enzymes appear to be responsive to alterations in glucose metabolized via the pentose phosphate pathway.     

Escherichia coli Virulence Protein NleH1 Interaction with the v-Crk Sarcoma Virus CT10 Oncogene-like Protein (CRKL) Governs NleH1 Inhibition of the Ribosomal Protein S3 (RPS3)/Nuclear Factor κB (NF-κB) Pathway

Enterohemorrhagic Escherichia coli and other attaching/effacing bacterial pathogens cause diarrhea in humans. These pathogens use a type III secretion system to inject virulence proteins (effectors) into host cells, some of which inhibit the innate immune system. The enterohemorrhagic E. coli NleH1 effector prevents the nuclear translocation of RPS3 (ribosomal protein S3) to inhibit its participation as a nuclear “specifier” of NF-κB binding to target gene promoters. NleH1 binds to RPS3 and inhibits its phosphorylation on Ser-209 by IκB kinase-β (IKKβ). However, the precise mechanism of this inhibition is unclear. NleH1 possesses a Ser/Thr protein kinase activity that is essential both for its ability to inhibit the RPS3/NF-κB pathway and for full virulence of the attaching/effacing mouse pathogen Citrobacter rodentium. However, neither RPS3 nor IKKβ is a substrate of NleH1 kinase activity. We therefore screened ∼9,000 human proteins to identify NleH1 kinase substrates and identified CRKL (v-Crk sarcoma virus CT10 oncogene-like protein), a substrate of the BCR/ABL kinase. Knockdown of CRKL abundance prevented NleH1 from inhibiting RPS3 nuclear translocation and NF-κB activity. CRKL residues Tyr-198 and Tyr-207 were required for interaction with NleH1. Lys-159, the kinase-active site of NleH1, was necessary for its interaction with CRKL. We also identified CRKL as an IKKβ interaction partner, mediated by CRKL Tyr-198. We propose that the CRKL interaction with IKKβ recruits NleH1 to the IKKβ complex, where NleH1 then inhibits the RPS3/NF-κB pathway.     

Huntingtin-associated Protein 1 (HAP1) Is a cGMP-dependent Kinase Anchoring Protein (GKAP) Specific for the cGMP-dependent Protein Kinase Iβ Isoform

Protein-protein interactions are important in providing compartmentalization and specificity in cellular signal transduction. Many studies have hallmarked the well designed compartmentalization of the cAMP-dependent protein kinase (PKA) through its anchoring proteins. Much less data are available on the compartmentalization of its closest homolog, cGMP-dependent protein kinase (PKG), via its own PKG anchoring proteins (GKAPs). For the enrichment, screening, and discovery of (novel) PKA anchoring proteins, a plethora of methodologies is available, including our previously described chemical proteomics approach based on immobilized cAMP or cGMP. Although this method was demonstrated to be effective, each immobilized cyclic nucleotide did not discriminate in the enrichment for either PKA or PKG and their secondary interactors. Hence, with PKG signaling components being less abundant in most tissues, it turned out to be challenging to enrich and identify GKAPs. Here we extend this cAMP-based chemical proteomics approach using competitive concentrations of free cyclic nucleotides to isolate each kinase and its secondary interactors. Using this approach, we identified Huntingtin-associated protein 1 (HAP1) as a putative novel GKAP. Through sequence alignment with known GKAPs and secondary structure prediction analysis, we defined a small sequence domain mediating the interaction with PKG Iβ but not PKG Iα. In vitro binding studies and site-directed mutagenesis further confirmed the specificity and affinity of HAP1 binding to the PKG Iβ N terminus. These data fully support that HAP1 is a GKAP, anchoring specifically to the cGMP-dependent protein kinase isoform Iβ, and provide further evidence that also PKG spatiotemporal signaling is largely controlled by anchoring proteins.     

Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) Regulates γ-Histone 2AX (γ-H2AX) Levels upon Ionizing Radiation

The microtubule-associated protein targeting protein for Xenopus kinesin-like protein 2 (TPX2) plays a key role in spindle assembly and is required for mitosis in human cells. In interphase, TPX2 is actively imported into the nucleus to prevent its premature activity in microtubule organization. To date, no function has been assigned to nuclear TPX2. We now report that TPX2 plays a role in the cellular response to DNA double strand breaks induced by ionizing radiation. Loss of TPX2 leads to inordinately strong and transient accumulation of ionizing radiation-dependent Ser-139-phosphorylated Histone 2AX (γ-H2AX) at G₀ and G₁ phases of the cell cycle. This is accompanied by the formation of increased numbers of high intensity γ-H2AX ionizing radiation-induced foci. Conversely, cells overexpressing TPX2 have reduced levels of γ-H2AX after ionizing radiation. Consistent with a role for TPX2 in the DNA damage response, we found that the protein accumulates at DNA double strand breaks and associates with the mediator of DNA damage checkpoint 1 (MDC1) and the ataxia telangiectasia mutated (ATM) kinase, both key regulators of γ-H2AX amplification. Pharmacologic inhibition or depletion of ATM or MDC1, but not of DNA-dependent protein kinase (DNA-PK), antagonizes the γ-H2AX phenotype caused by TPX2 depletion. Importantly, the regulation of γ-H2AX signals by TPX2 is not associated with apoptosis or the mitotic functions of TPX2. In sum, our study identifies a novel and the first nuclear function for TPX2 in the cellular responses to DNA damage.     

14-3-3ζ Protein Regulates Anterograde Transport of the Human κ-Opioid Receptor (hKOPR)

By proteomic analysis, we found that 14-3-3ζ was one of the proteins co-immunoprecipitated with human κ-opioid receptor (hKOPR) from extracts of solubilized Neuro2A cells stably expressing FLAG-hKOPR (N2A-FLAG-hKOPR cells). 14-3-3 proteins are a family of conserved regulatory molecules in eukaryotic cells, where they participate in signal transduction, metabolism, and membrane protein transport. 14-3-3ζ co-localized with the hKOPR in N2A cells. The hKOPR C-tail interacted with 14-3-3ζ in rat brain extracts and bound directly to purified 14-3-3ζ as demonstrated by pulldown techniques. 14-3-3ζ siRNA decreased expression of the hKOPR in N2A-FLAG-hKOPR cells and cultured primary cortical neurons of E19 rats by ∼25% as determined by immunoblotting, ligand binding, and flow cytometry. The effect of 14-3-3ζ siRNA was reversed by overexpression of 14-3-3ζ. Expression of the 14-3-3 scavenger protein pGpLI-R18 also decreased hKOPR expression. 14-3-3ζ siRNA did not change expressions of the hDOPR and rMOPR in N2A cells. Pulse-chase study showed that 14-3-3ζ siRNA decreased the amount of mature hKOPR but did not change the rate of maturation or stability of hKOPR protein. Mutations of R354A/S358A in the putative 14-3-3 interaction motif ³⁵⁴RQSTS³⁵⁸ in the hKOPR C-tail reduced interaction of the hKOPR with 14-3-3ζ and abolished the effect of 14-3-3ζ knockdown on hKOPR expression. Mutation of the endoplasmic reticulum retention motif ³⁵⁹RVR adjacent to the 14-3-3 interaction motif in the hKOPR C-tail decreased interaction of coatomer protein I (COPI) with the hKOPR and abolished 14-3-3ζ-mediated regulation of hKOPR expression. 14-3-3ζ knockdown increased association of COPI with the hKOPR. These results suggest that 14-3-3ζ promotes expression of the hKOPR by inhibiting COPI and RVR motif-mediated endoplasmic reticulum localization machinery.     

Integration of G Protein α (Gα) Signaling by the Regulator of G Protein Signaling 14 (RGS14)

RGS14 contains distinct binding sites for both active (GTP-bound) and inactive (GDP-bound) forms of Gα subunits. The N-terminal regulator of G protein signaling (RGS) domain binds active Gα_i/o-GTP, whereas the C-terminal G protein regulatory (GPR) motif binds inactive Gα_i1/3-GDP. The molecular basis for how RGS14 binds different activation states of Gα proteins to integrate G protein signaling is unknown. Here we explored the intramolecular communication between the GPR motif and the RGS domain upon G protein binding and examined whether RGS14 can functionally interact with two distinct forms of Gα subunits simultaneously. Using complementary cellular and biochemical approaches, we demonstrate that RGS14 forms a stable complex with inactive Gα_i1-GDP at the plasma membrane and that free cytosolic RGS14 is recruited to the plasma membrane by activated Gα_o-AlF₄⁻. Bioluminescence resonance energy transfer studies showed that RGS14 adopts different conformations in live cells when bound to Gα in different activation states. Hydrogen/deuterium exchange mass spectrometry revealed that RGS14 is a very dynamic protein that undergoes allosteric conformational changes when inactive Gα_i1-GDP binds the GPR motif. Pure RGS14 forms a ternary complex with Gα_o-AlF₄⁻ and an AlF₄⁻-insensitive mutant (G42R) of Gα_i1-GDP, as observed by size exclusion chromatography and differential hydrogen/deuterium exchange. Finally, a preformed RGS14·Gα_i1-GDP complex exhibits full capacity to stimulate the GTPase activity of Gα_o-GTP, demonstrating that RGS14 can functionally engage two distinct forms of Gα subunits simultaneously. Based on these findings, we propose a working model for how RGS14 integrates multiple G protein signals in host CA2 hippocampal neurons to modulate synaptic plasticity.     

Residues 240–250 in the C-Terminus of the Pirh2 Protein Complement the Function of the RING Domain in Self-Ubiquitination of the Pirh2 Protein

Pirh2 is a p53 inducible gene that encodes a RING-H2 domain and is proposed to be a main regulator of p53 protein, thus fine tuning the DNA damage response. Pirh2 interacts physically with p53 and promotes its MDM2-independent ubiquitination and subsequent degradation as well as participates in an auto-regulatory feedback loop that controls p53 function. Pirh2 also self-ubiquitinates. Interestingly, Pirh2 is overexpressed in a wide range of human tumors. In this study, we investigated the domains and residues essential for Pirh2 self-ubiquitination. Deletions were made in each of the three major domains of Pirh2: the N-terminal domain (NTD), Ring domain (RING), and C-terminal domain (CTD). The effects of these deletions on Pirh2 self-ubiquitination were then assessed using in vitro ubiquitination assays. Our results demonstrate that the RING domain is essential, but not sufficient, for Pirh2 self-ubiquitination and that residues 240–250 of the C-terminal domain are also essential. Our results demonstrate that Pirh2 mediated p53 polyubiquitination occurs mainly through the K48 residue of ubiquitin in vitro. Our data further our understanding of the mechanism of Pirh2 self-ubiquitination and may help identify valuable therapeutic targets that play roles in reducing the effects of the overexpression of Pirh2, thus maximizing p53''s response to DNA damage.     

Synthesis and characterization of [Pt(COD)Cl(NO3)] and [Pt(COD)(NO3)2] and the use of [Pt(COD)Clx(NO3)2−x] in the preparation of cis[Pt(PPh3)2Clx(NO3)2−x] (x=0, 1, 2) and [Pt(PPh3)3L](NO3) (L=Cl,NO3)

[Pt(COD)Cl₂] (COD=1,5-cyclooctadiene) is a versatile starting material for the synthesis of Pt(II) compounds. The preparations of the new compounds [Pt(COD)Cl(NO₃)], [Pt(COD)(NO₃)₂] and [Pt(PPh₃)₃(NO₃)](NO₃) and also of the known compounds cis[Pt(PPh₃)₂Cl₂], cis [Pt(PPh₃)₂Cl(NO₃)], cis[Pt(PPh₃)₂(NO₃)₂] and [Pt(PPh₃)₃Cl](NO₃)are reported. The compounds are characterized by elemental analysis, ³¹P{¹H} NMR spectroscopy and IR spectroscopy.     

Soybean PM2 Protein (LEA3) Confers the Tolerance of Escherichia coli and Stabilization of Enzyme Activity Under Diverse Stresses

Late embryogenesis abundant (LEA) proteins are closely associated with the tolerance of diverse stresses in organisms. To elucidate the function of group 3 LEA proteins, the soybean PM2 protein (LEA3) was expressed in E. coli and the protective function of the PM2 protein was assayed both in vivo and in vitro. The results of a spot assay and survival ratio demonstrated that the expression of the PM2 protein conferred the tolerance to the E. coli recombinant for different temperature conditions (4, −20 or 50°C) or high-salinity stresses (120 mmol/l MgCl₂ or 120 mmol/l CaCl₂). In addition, it was demonstrated that the in vitro addition of the PM2 protein could prevent the lactate dehydrogenase (LDH) inactivation normally induced by freeze–thaw. In the 62°C condition, the PM2 protein (1:5 mass ratio to LDH) effectively prevented the LDH thermo-denaturation by acting synergistically with trehalose (62.5 μg/ml), although the PM2 protein alone at this concentration showed little protective effect on LDH activity. Furthermore, the results showed that the PM2 protein could partially prevent the thermo-denaturation of the bacterial proteome after boiling for 2 min. Based on these results, we propose that the PM2 protein itself, or together with trehalose, conferred the tolerance to the E. coli recombinant against diverse stresses by protecting proteins and enzyme activity under low- or high- temperature conditions.     

Regulation of the Src Homology 2 Domain-containing Inositol 5��-Phosphatase (SHIP1) by the Cyclic AMP-dependent Protein Kinase

Many agents that activate hematopoietic cells use phos pha tidyl ino si tol 3,4,5-trisphosphate (PtdIns 3,4,5-P₃) to initiate signaling cascades. The SH2 domain-containing inositol 5′ phosphatase, SHIP1, regulates hematopoietic cell function by opposing the action of phos pha tidyl ino si tol 3-kinase and reducing the levels of PtdIns 3,4,5-P₃. Activation of the cyclic AMP-de pend ent protein kinase (PKA) also opposes many of the pro-inflammatory responses of hematopoietic cells. We tested to see whether the activity of SHIP1 was regulated via phos pho ryl a tion with PKA. We prepared pure recombinant SHIP1 from HEK-293 cells and found it can be rapidly phos pho ryl a ted by PKA to a stoichiometry of 0.6 mol of PO₄/mol of SHIP1. In ³²P-labeled HEK-293 cells transfected with SHIP1, stimulation with Sp-adenosine 3′,5′-cyclic monophosphorothioate triethylammonium salt hydrate (Sp-cAMPS) or activation of the β-adrenergic receptor increased the phos pho ryl a tion state of SHIP1. Inhibition of protein phosphatase activity with okadaic acid also increased the phos pho ryl a tion of SHIP1. Phosphorylation of SHIP1 in vitro or in cells by PKA increased the 5′ phosphatase activity of SHIP1 by 2–3-fold. Elevation of Ca²⁺ in DT40 cells in response to B cell receptor cross-linking, an indicator of PtdIns 3,4,5-P₃ levels, was markedly blunted by pretreatment with Sp-cAMPS. This effect was absent in SHIP^−/− DT40 cells showing that the effect of Sp-cAMPS in DT40 cells is SHIP1-de pend ent. Sp-cAMPS also blunted the ability of the B cell receptor to increase the phos pho ryl a tion of Akt in DT40 and A20 cells. Overall, activation of G protein-coupled receptors that raise cyclic AMP cause SHIP1 to be phos pho ryl a ted and stimulate its inositol phosphatase activity. These results outline a novel mechanism of SHIP1 regulation.Activation of phosphatidylinositol 3-kinase (PtdIns 3-kinase)² is central to regulation of multiple cell functions including cell shape changes, cell migration, cell activation, and proliferation (1). PtdIns 3-kinase phosphorylates phosphatidylinositol 4,5-bisphosphate in the inner leaflet of the plasma membrane to generate phosphatidylinositol 3,4,5-trisphosphate (PtdIns 3,4,5-P₃) (2). PtdIns 3,4,5-P₃ then activates downstream signaling pathways by interacting with pleckstrin homology domain-containing proteins, such as phosphoinositide-dependent kinase 1 and the serine-threonine kinase Akt (3). The finding of abnormal activation of the PtdIns 3-kinase pathway in cancer cells has led to interest in the development of inhibitors for PtdIns 3-kinase (4).The level of PtdIns 3,4,5-P₃ is stimulated by multiple members of the PtdIns 3-kinase family (2) and is opposed by two phosphatidylinositol phosphatases: the Src homology 2 (SH2) domain-containing inositol 5′ phosphatase (SHIP) and the 3′ inositol phosphatase, phosphatase and tensin homolog (PTEN) (5). PTEN removes phosphate from the 3′ position in the inositol ring of PtdIns 3,4,5-P₃ and converts it to phosphatidylinositol 4,5-bisphosphate (6). PTEN has a C2 domain, a PDZ-binding motif, and a N-terminal phosphatidylinositol 4,5-bisphosphate binding motif essential for translocation to the membrane and interaction with other regulatory proteins (7). There are serine and threonine residues in PTEN that have been found to be phosphorylated, but their role in regulating the activity of the enzyme is not clear (8). Mutations in the PTEN protein have been observed in many tumors, suggesting a role for this enzyme in cancer (9).In contrast, SHIP dephosphorylates the 5′ position on the inositol ring and produces phosphatidylinositol 3,4-bisphosphate (10). There are three isoforms of SHIP: the 145-kDa hematopoietic cell restricted SHIP (also known as SHIP1); the 104-kDa stem cell-restricted SHIP, sSHIP; and the more widely expressed 150-kDa SHIP2 (11). SHIP1 is the major inositol phosphatase regulating PtdIns 3,4,5-P₃ in monocytes, macrophages, B cells, and T cells (11). SHIP1 has three known structural features: the N-terminal SH2 domain, the central inositol 5′ phosphatase domain, and two NPXY sequences in the C-terminal region. The currently accepted model for regulation of PtdIns 3,4,5-P₃ levels by SHIP1 envisions translocation of SHIP1 from the cytosol to the membrane. Upon stimulation by growth factors, cytokine receptors, or immunoreceptors, SHIP1 is recruited via its N-terminal SH2 domain to phosphorylated tyrosine residues in receptor kinases and degrades the elevated levels of PtdIns 3,4,5-P₃ near the activated receptor (12). During this translocation process, SHIP1 is not thought to change its 5′ phosphatase activity (13). Although it is known that SHIP1 can be phosphorylated on tyrosine residues by the lyn cytoplasmic kinase (12) or following the activation of the T cell receptor (14), neither event appears to influence the 5′ phosphatase activity. To date, direct regulation of SHIP1 activity by serine/threonine kinases has not been studied.Activation of G protein-coupled receptors that raise cAMP (i.e. β-adrenergic receptors or adenosine A_2a receptors) is known to blunt the pro-inflammatory responses generated by receptors that raise the level of PtdIns 3,4,5-P₃ (15). Therefore, we investigated the possibility that phosphorylation of SHIP1 by cyclic AMP-dependent protein kinase (PKA) might regulate the activity of SHIP1. We found that SHIP1 can be phosphorylated by PKA both in vitro and in cells leading to a stimulation of SHIP1 activity. Activation of PKA in DT40 and A20 cells blunted indicators of the PtdIns 3,4,5-P₃ response to B cell receptor stimulation. These results indicate that SHIP1 activity can be regulated both in vitro and in cells by activation of the cyclic AMP-dependent protein kinase and highlight a new mode of SHIP regulation by G protein-coupled receptors.