Serine/threonine phosphorylation regulates binding of C hnRNP proteins to pre-mRNA.

The C hnRNP proteins bind to nascent pre-mRNA and are thought to participate in an early step of the pre-mRNA splicing pathway. We report here that C hnRNP proteins are phosphorylated by a casein kinase II activity in a HeLa cell nuclear extract and that dephosphorylation of C hnRNP proteins is inhibited by the specific protein-serine/threonine-phosphatase 1/2A inhibitor okadaic acid. We further find that dephosphorylation of C hnRNP proteins is required for their binding to adenovirus and human beta-globin pre-mRNAs. These results indicate that the participation of C hnRNP proteins in pre-spliceosome assembly is coupled to a dynamic cycle of their phosphorylation and dephosphorylation.     

cAMP-dependent protein kinase phosphorylation of the acid-sensing ion channel-1 regulates its binding to the protein interacting with C-kinase-1

The acid-sensing ion channel-1 (ASIC1) contributes to synaptic plasticity and may influence the response to cerebral ischemia and acidosis. We found that cAMP-dependent protein kinase phosphorylated heterologously expressed ASIC1 and endogenous ASIC1 in brain slices. ASIC1 also showed significant phosphorylation under basal conditions. Previous studies showed that the extreme C-terminal residues of ASIC1 bind the PDZ domain of the protein interacting with C-kinase-1 (PICK1). We found that protein kinase A phosphorylation of Ser-479 in the ASIC1 C terminus interfered with PICK1 binding. In contrast, minimizing phosphorylation or mutating Ser-479 to Ala enhanced PICK1 binding. Phosphorylation-dependent disruption of PICK1 binding reduced the cellular colocalization of ASIC1 and PICK1. Thus, the ASIC1 C terminus contains two sites that influence its binding to PICK1. Regulation of this interaction by phosphorylation provides a mechanism to control the cellular localization of ASIC1.     

Pink1 regulates the oxidative phosphorylation machinery via mitochondrial fission

Mutations in PTEN-induced kinase 1 (PINK1), a mitochondrial Ser/Thr kinase, cause an autosomal recessive form of Parkinson''s disease (PD), PARK6. To investigate the mechanism of PINK1 pathogenesis, we used the Drosophila Pink1 knockout (KO) model. In mitochondria isolated from Pink1-KO flies, mitochondrial respiration driven by the electron transport chain (ETC) is significantly reduced. This reduction is the result of a decrease in ETC complex I and IV enzymatic activity. As a consequence, Pink1-KO flies also display a reduced mitochondrial ATP synthesis. Because mitochondrial dynamics is important for mitochondrial function and Pink1-KO flies have defects in mitochondrial fission, we explored whether fission machinery deficits underlie the bioenergetic defect in Pink1-KO flies. We found that the bioenergetic defects in the Pink1-KO can be ameliorated by expression of Drp1, a key molecule in mitochondrial fission. Further investigation of the ETC complex integrity in wild type, Pink1-KO, PInk1-KO/Drp1 transgenic, or Drp1 transgenic flies indicates that the reduced ETC complex activity is likely derived from a defect in the ETC complex assembly, which can be partially rescued by increasing mitochondrial fission. Taken together, these results suggest a unique pathogenic mechanism of PINK1 PD: The loss of PINK1 impairs mitochondrial fission, which causes defective assembly of the ETC complexes, leading to abnormal bioenergetics.     

Pink1 regulates mitochondrial dynamics through interaction with the fission/fusion machinery

Mitochondria form dynamic tubular networks that undergo frequent morphological changes through fission and fusion, the imbalance of which can affect cell survival in general and impact synaptic transmission and plasticity in neurons in particular. Some core components of the mitochondrial fission/fusion machinery, including the dynamin-like GTPases Drp1, Mitofusin, Opa1, and the Drp1-interacting protein Fis1, have been identified. How the fission and fusion processes are regulated under normal conditions and the extent to which defects in mitochondrial fission/fusion are involved in various disease conditions are poorly understood. Mitochondrial malfunction tends to cause diseases with brain and skeletal muscle manifestations and has been implicated in neurodegenerative diseases such as Parkinson's disease (PD). Whether abnormal mitochondrial fission or fusion plays a role in PD pathogenesis has not been shown. Here, we show that Pink1, a mitochondria-targeted Ser/Thr kinase linked to familial PD, genetically interacts with the mitochondrial fission/fusion machinery and modulates mitochondrial dynamics. Genetic manipulations that promote mitochondrial fission suppress Drosophila Pink1 mutant phenotypes in indirect flight muscle and dopamine neurons, whereas decreased fission has opposite effects. In Drosophila and mammalian cells, overexpression of Pink1 promotes mitochondrial fission, whereas inhibition of Pink1 leads to excessive fusion. Our genetic interaction results suggest that Fis1 may act in-between Pink1 and Drp1 in controlling mitochondrial fission. These results reveal a cell biological role for Pink1 and establish mitochondrial fission/fusion as a paradigm for PD research. Compounds that modulate mitochondrial fission/fusion could have therapeutic value in PD intervention.     

Sphingosine kinase 1 regulates mucin production via ERK phosphorylation

Our previous report showed that inhibition of sphingosine kinase (SphK) ameliorates eosinophilic inflammation and mucin production in a mouse asthmatic model. To clarify the role of SphK in airway mucin production, we utilized the mouse asthmatic model and found that both SphK and MUC5AC expression were increased and co-localized in airway epithelium. Next we cultured normal human bronchial epithelial cells in an air–liquid interface and treated with IL-13 to induce their differentiation into goblet cells. We found that SphK1 and MUC5AC expression was increased by IL-13 treatment at both protein and mRNA levels, whereas SphK2 expression was not changed. N,N-dimethylsphingosine (DMS), a potent SphK inhibitor, decreased MUC5AC expression up-regulated by IL-13 treatment. Furthermore, DMS inhibited IL-13-induced ERK1/2 phosphorylation but neither p38 MAPK nor STAT6 phosphorylation. These results suggest that SphK1 is involved in MUC5AC production induced by IL-13 upstream of ERK1/2 phosphorylation, and independent of STAT6 phosphorylation.     

Structural characterization of the Get4/Get5 complex and its interaction with Get3

The recently elucidated Get proteins are responsible for the targeted delivery of the majority of tail-anchored (TA) proteins to the endoplasmic reticulum. Get4 and Get5 have been identified in the early steps of the pathway mediating TA substrate delivery to the cytoplasmic targeting factor Get3. Here we report a crystal structure of Get4 and an N-terminal fragment of Get5 from Saccharomyces cerevisae. We show Get4 and Get5 (Get4/5) form an intimate complex that exists as a dimer (two copies of Get4/5) mediated by the C-terminus of Get5. We further demonstrate that Get3 specifically binds to a conserved surface on Get4 in a nucleotide dependent manner. This work provides further evidence for a model in which Get4/5 operates upstream of Get3 and mediates the specific delivery of a TA substrate.     

Protein phosphatase 1 regulates the phosphorylation state of the polarity scaffold Par-3

Phosphorylation of the polarity protein Par-3 by the serine/threonine kinases aPKCζ/ι and Par-1 (EMK1/MARK2) regulates various aspects of epithelial cell polarity, but little is known about the mechanisms by which these posttranslational modifications are reversed. We find that the serine/threonine protein phosphatase PP1 (predominantly the α isoform) binds Par-3, which localizes to tight junctions in MDCKII cells. PP1α can associate with multiple sites on Par-3 while retaining its phosphatase activity. By using a quantitative mass spectrometry-based technique, multiple reaction monitoring, we show that PP1α specifically dephosphorylates Ser-144 and Ser-824 of mouse Par-3, as well as a peptide encompassing Ser-885. Consistent with these observations, PP1α regulates the binding of 14-3-3 proteins and the atypical protein kinase C (aPKC) ζ to Par-3. Furthermore, the induced expression of a catalytically inactive mutant of PP1α severely delays the formation of functional tight junctions in MDCKII cells. Collectively, these results show that Par-3 functions as a scaffold, coordinating both serine/threonine kinases and the PP1α phosphatase, thereby providing dynamic control of the phosphorylation events that regulate the Par-3/aPKC complex.     

Structural characterization of the interaction of Ubp6 with the 26S proteasome

In eukaryotic cells, the 26S proteasome is responsible for the regulated degradation of intracellular proteins. Several cofactors interact transiently with this large macromolecular machine and modulate its function. The deubiquitylating enzyme ubiquitin C-terminal hydrolase 6 [Ubp6; ubiquitin-specific protease (USP) 14 in mammals] is the most abundant proteasome-interacting protein and has multiple roles in regulating proteasome function. Here, we investigate the structural basis of the interaction between Ubp6 and the 26S proteasome in the presence and absence of the inhibitor ubiquitin aldehyde. To this end we have used single-particle electron cryomicroscopy in combination with cross-linking and mass spectrometry. Ubp6 binds to the regulatory particle non-ATPase (Rpn) 1 via its N-terminal ubiquitin-like domain, whereas its catalytic USP domain is positioned variably. Addition of ubiquitin aldehyde stabilizes the binding of the USP domain in a position where it bridges the proteasome subunits Rpn1 and the regulatory particle triple-A ATPase (Rpt) 1. The USP domain binds to Rpt1 in the immediate vicinity of the Ubp6 active site, which may effect its activation. The catalytic triad is positioned in proximity to the mouth of the ATPase module and to the deubiquitylating enzyme Rpn11, strongly implying their functional linkage. On the proteasome side, binding of Ubp6 favors conformational switching of the 26S proteasome into an intermediate-energy conformational state, in particular upon the addition of ubiquitin aldehyde. This modulation of the conformational space of the 26S proteasome by Ubp6 explains the effects of Ubp6 on the kinetics of proteasomal degradation.Degradation of proteins that are misfolded, damaged, or no longer needed is an essential element of cellular homeostasis. In eukaryotic cells, the ubiquitin-proteasome system (UPS) is the major pathway for regulated protein degradation (1). Proteins that are processed by the UPS are marked for destruction by polyubiquitin chains, which are recognized as a degradation signal by the 26S proteasome.The 26S proteasome consists of the core particle (CP), which degrades substrates into short peptides, and one or two 19S regulatory particles (RP), which associate with the ends of the cylinder-shaped CP to recruit substrates and prepare them for degradation (2, 3). Although the structure of the CP has been known for more than two decades (4, 5), the molecular architecture of the RP was unraveled by cryo-electron microscope (EM)–based approaches only recently (6–9). It comprises six RP triple A (AAA) ATPases (Rpt), 1–6, and 13 RP non-ATPases (Rpn), 1–3, 5–13, and 15. Similar to AAA-ATPases in prokaryotic ATP-dependent proteases, the Rpts form a hexameric ring that binds to the ends of the CP and is responsible for substrate unfolding and translocation into the CP. Unlike their prokaryotic counterparts, the Rpts are surrounded by non-ATPases. Apart from Rpn1, all Rpns form a cohesive structure, which places the RP’s catalytic subunits at their optimal positions for efficient proteasomal degradation. The deubiquitylating enzyme (DUB) Rpn11, which is responsible for the removal of polyubiquitin chains from substrates before degradation (10, 11), is positioned near the oligosaccharide-binding domain (OB) ring forming the mouth of the AAA-ATPase. The resident receptors for ubiquitin chains, Rpn10 and Rpn13, are positioned at the distal ends of the RP (12).Recent cryo-EM analyses revealed the conformational plasticity of the RP. At least three distinct states, which we refer to as “s1–s3,” can be distinguished (13). In ATP-containing buffer, purified 26S proteasomes primarily adopt the s1 state, which is characterized by pronounced off-axis positioning of the AAA-ATPase hexamer with respect to the CP and a staircase arrangement of the Rpts with Rpt3 in the most elevated position (6–9). Under the same conditions a minority of particles (∼20%) adopts the s2 state, in which the axis of the AAA-ATPase is positioned closer to that of the CP and the Rpns concomitantly rotate largely en bloc by ∼25° (13). As a consequence of this Rpn motion, the active site of Rpn11 becomes accessible to the polyubiquitin chain of the substrate, and the ubiquitin receptor Rpn10 is positioned closer to the AAA-ATPase module. A third conformation, s3, was found in the presence of the slowly hydrolysable ATP analogue ATP-γS (14) or upon the addition of polyubiquitylated substrate to 26S proteasomes with dysfunctional Rpn11 (15). Characteristics of the s3 state are a changed staircase arrangement of the AAA-module with Rpt1 most elevated and a further translation of the Rpns compared with s2, leaving Rpn11 and Rpn10 essentially invariant with respect to the mouth of the AAA-ATPase module.Proteasome function is modulated by transiently binding cofactors, the proteasome-interacting proteins (PIPs) (16–19), which typically are found in substoichiometric amounts in purified proteasomes (20). Of these, ubiquitin C-terminal hydrolase 6 [Ubp6, human ubiquitin-specific protease 14 (Usp14)] is most abundant. It consists of an N-terminal ubiquitin-like (UBL) domain, which associates primarily with the RP via Rpn1 (21, 22), a disordered linker of ∼25 residues, and an ubiquitin-specific protease (USP) domain. The DUB activity of Ubp6 is low in isolation but increases dramatically upon binding to the 26S proteasome (16, 23–25). One main function of Ubp6 seems to be to delay the degradation of polyubiquitylated substrates by progressively deubiquitylating them (25, 26). Ubp6 thus seems to serve as a timing device. A pharmacological agent that inhibits deubiquitylation by the human Ubp6 homolog Usp14, enhances the chance that the protein is degraded (26). Consequently, Usp14 is an attractive drug target, e.g., to prevent the accumulation of protein aggregates associated with neurodegenerative diseases. Interestingly, the catalytic activity is not required for inhibiting the degradation of folded substrates (17, 25), but the structural basis is unclear. On the other hand, binding of Ub-conjugates or the USP inhibitor ubiquitin-aldehyde (UbAld) to proteasome-bound Ubp6 enhances the degradation of short unfolded peptides (27) and activates the proteasomal ATPases (28), both indicating a conformational change of the 26S proteasome. To gain structural insights into the mechanisms of the proteasomal regulation by Ubp6, we determined the position of Ubp6 in complex with UbAld bound to the 26S proteasome and its consequences for the conformation of the RP.     

SDF-1/CXCR4 and VLA-4 interaction regulates homing in Waldenstrom macroglobulinemia

Waldenstrom macroglobulinemia (WM) is characterized by widespread involvement of the bone marrow at the time of diagnosis, implying continuous homing of WM cells into the marrow. The mechanisms by which trafficking of the malignant cells into the bone marrow has not been previously elucidated. In this study, we show that WM cells express high levels of chemokine and adhesion receptors, including CXCR4 and VLA-4. We showed that CXCR4 was essential for the migration and trans-endothelial migration of WM cells under static and dynamic shear flow conditions, with significant inhibition of migration using CXCR4 knockdown or the CXCR4 inhibitor AMD3100. Similarly, CXCR4 or VLA-4 inhibition led to significant inhibition of adhesion to fibronectin, stromal cells, and endothelial cells. Decreased adhesion of WM cells to stromal cells by AMD3100 led to increased sensitivity of these cells to cytotoxicity by bortezomib. To further investigate the mechanisms of CXCR4-dependent adhesion, we showed that CXCR4 and VLA-4 directly interact in response to SDF-1, we further investigated downstream signaling pathways regulating migration and adhesion in WM. Together, these studies demonstrate that the CXCR4/SDF-1 axis interacts with VLA-4 in regulating migration and adhesion of WM cells in the bone marrow microenvironment.     

USP1 regulates AKT phosphorylation by modulating the stability of PHLPP1 in lung cancer cells

Background

Hyperactivation of phosphatidylinositol 3-kinase/Akt signaling is commonly associated with human tumors including lung cancers. PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), which terminates Akt signaling by directly dephosphorylating and inactivating Akt, has been identified as a tumor suppressor. The protein level of PHLPP1 is regulated by E3 ligase beta-TRCP, however, the deubiquitinase for PHLPP1 is still not known.

Methods

The mRNA levels of USP1 and PHLPP1 in lung cancer cells and tissues were determined by real-time PCR. The half-life of PHLPP1 was detected by CHX assay. The interaction between USP1 and PHLPP1 was examined by immunoprecipitation and GST pull-down assay.

Results

Both USP1 and PHLPP1 are low expressed in lung cancer cells and tissues and silencing of USP1 by RNA interference significantly decreased the half-life of PHLPP1, which in turn amplified Akt1 phosphorylation. Our data identified a novel USP1-PHLPP1-Akt signaling axis, and decreased USP1 level in lung cancer cells may play an important role in lung cancer progress.     

A protein phosphorylation/dephosphorylation network regulates a plant potassium channel

Potassium (K(+)) is an essential nutrient for plant growth and development. Plants often adapt to low K(+) conditions by increasing their K(+) uptake capability. Recent studies have led to the identification of a calcium signaling pathway that enables plants to act in this capacity. Calcium is linked to two calcineurin B-like calcium sensors (CBLs) and a target kinase (CBL-interacting protein kinase 23 or CIPK23) that, in turn, appears to phosphorylate and activate the potassium channel, Arabidopsis K(+) transporter 1 (AKT1), responsible for K(+) uptake in roots. Here, we report evidence that this regulatory mechanism is more elaborate than earlier envisaged. The recently described pathway is part of an extensive network whereby several CBLs interact with multiple CIPKs in the activation of the potassium channel, AKT1. The physical interactions among the CBL, CIPK, and AKT1 components provide a mechanism for specifying the members of the CBL and CIPK families functional in AKT1 regulation. The interaction between the CIPKs and AKT1 was found to involve the kinase domain of the CIPK component and the ankyrin repeat domain of the channel. Furthermore, we identified a 2C-type protein phosphatase that physically interacts and inactivates the AKT1 channel. These findings provide evidence that the calcium-sensitive CBL and CIPK families together with 2C-type protein phosphatases form a protein phoshporylation/dephosphorylation network that regulates the AKT1 channel for K(+) transport in plants.