Attenuation of T Cell Receptor Signaling by Serine Phosphorylation-mediated Lysine 30 Ubiquitination of SLP-76 Protein

SLP-76 (SH2 domain-containing leukocyte protein of 76 kDa) is an adaptor protein that is essential for T cell development and T cell receptor (TCR) signaling activation. Previous studies have identified an important negative feedback regulation of SLP-76 by HPK1 (hematopoietic progenitor kinase 1; MAP4K1)-induced Ser-376 phosphorylation. Ser-376 phosphorylation of SLP-76 mediates 14-3-3 binding, resulting in the attenuation of SLP-76 activation and downstream signaling; however, the underlying mechanism of this action remains unknown. Here, we report that phosphorylated SLP-76 is ubiquitinated and targeted for proteasomal degradation during TCR signaling. SLP-76 ubiquitination is mediated by Ser-376 phosphorylation. Furthermore, Lys-30 is identified as a ubiquitination site of SLP-76. Loss of Lys-30 ubiquitination of SLP-76 results in enhanced anti-CD3 antibody-induced ERK and JNK activation. These results reveal a novel regulation mechanism of SLP-76 by ubiquitination and proteasomal degradation of activated SLP-76, which is mediated by Ser-376 phosphorylation, leading to down-regulation of TCR signaling.     

WNK4 Kinase Stimulates Caveola-mediated Endocytosis of TRPV5 Amplifying the Dynamic Range of Regulation of the Channel by Protein Kinase C

WNK4 (with-no-lysine (K) kinase-4) is present in the distal nephron of the kidney and plays an important role in the regulation of renal ion transport. The epithelial Ca²⁺ channel TRPV5 (transient receptor potential vanilloid 5) is the gatekeeper of transcellular Ca²⁺ reabsorption in the distal nephron. Previously, we reported that activation of protein kinase C (PKC) increases cell-surface abundance of TRPV5 by inhibiting caveola-mediated endocytosis of the channel. Here, we report that WNK4 decreases cell-surface abundance of TRPV5 by enhancing its endocytosis. Deletion analysis revealed that stimulation of endocytosis of TRPV5 involves amino acids outside the kinase domain of WNK4. We also investigated interplay between WNK4 and PKC regulation of TRPV5. The maximal level of TRPV5 current density stimulated by the PKC activator 1-oleoyl-acetyl-sn-glycerol (OAG) is the same with or without WNK4. The relative increase of TRPV5 current stimulated by OAG, however, is greater in the presence of WNK4 compared with that without WNK4 (∼215% increase versus 60% increase above the level without OAG). Moreover, the rate of increase of TRPV5 by OAG is faster with WNK4 than without WNK4. The enhanced increase of TRPV5 in the presence of WNK4 is also observed when PKC is activated by parathyroid hormones. Thus, WNK4 exerts tonic inhibition of TRPV5 by stimulating caveola-mediated endocytosis. The lower basal TRPV5 level in the presence of WNK4 allows amplification of the stimulation of channel by PKC. This interaction between WNK4 and PKC regulation of TRPV5 may be important for physiological regulation of renal Ca²⁺ reabsorption by parathyroid hormones or the tissue kallikrein in vivo.     

Ankyrin Repeat Domain Protein 2 and Inhibitor of DNA Binding 3 Cooperatively Inhibit Myoblast Differentiation by Physical Interaction

Ankyrin repeat domain protein 2 (ANKRD2) translocates from the nucleus to the cytoplasm upon myogenic induction. Overexpression of ANKRD2 inhibits C2C12 myoblast differentiation. However, the mechanism by which ANKRD2 inhibits myoblast differentiation is unknown. We demonstrate that the primary myoblasts of mdm (muscular dystrophy with myositis) mice (pMB^mdm) overexpress ANKRD2 and ID3 (inhibitor of DNA binding 3) proteins and are unable to differentiate into myotubes upon myogenic induction. Although suppression of either ANKRD2 or ID3 induces myoblast differentiation in mdm mice, overexpression of ANKRD2 and inhibition of ID3 or vice versa is insufficient to inhibit myoblast differentiation in WT mice. We identified that ANKRD2 and ID3 cooperatively inhibit myoblast differentiation by physical interaction. Interestingly, although MyoD activates the Ankrd2 promoter in the skeletal muscles of wild-type mice, SREBP-1 (sterol regulatory element binding protein-1) activates the same promoter in the skeletal muscles of mdm mice, suggesting the differential regulation of Ankrd2. Overall, we uncovered a novel pathway in which SREBP-1/ANKRD2/ID3 activation inhibits myoblast differentiation, and we propose that this pathway acts as a critical determinant of the skeletal muscle developmental program.     

Osx transcriptional regulation is mediated by additional pathways to BMP2/Smad signaling

Bone morphogenetic protein (BMP)-2 induces Osterix (Osx) in mouse C2C12 cells and chondrocytes. Genetic studies place Osx downstream to the BMP-2/Smad/Runx2 signaling pathway; however, limited information is available on the mediators of Osx expression in osteoblast lineage commitment. Several lines of research implicate the presence of Runx2-independent ossification. Therefore, the purpose of this study was to identify possible mediators of Osx expression beyond the BMP-2/Smad pathway. Using real-time RT-PCR, we showed upregulation of Osx in response to BMP-2 in human mesenchymal stem cells (hMSC). Insulin-like growth factor (IGF)-I upregulated Osx, but not Runx2. Further, IGF-I in combination with BMP-2 was synergistic for Osx, suggesting a pathway beyond Smad signaling. MAPK was tested as a common mediator across BMP-2 and IGF-I signaling pathways. Inhibition of MAPK component ERK1/2 did not affect Runx2 gene expression, but inhibited Osx expression and matrix mineralization. BMP-2-mediated Osx expression was downregulated in response to p38 inhibition. We therefore conclude that during osteogenic lineage progression, in addition to the BMP-2/Smad pathway, IGF-I and MAPK signaling may mediate Osx.     

Direct Association of Sprouty-related Protein with an EVH1 Domain (SPRED) 1 or SPRED2 with DYRK1A Modifies Substrate/Kinase Interactions

The mammalian SPRED (Sprouty-related protein with an EVH1 domain) proteins include a family of three members, SPRED1–3. Currently, little is known about their biochemistry. The best described, SPRED1, has been shown to inhibit the Ras/ERK pathway downstream of Ras. All three SPREDs have a cysteine-rich domain (CRD) that has high homology to the CRD of the Sprouty family of proteins, several of which are also Ras/ERK inhibitors. In the belief that binding partners would clarify SPRED function, we assayed for their associated proteins. Here, we describe the direct and endogenous interaction of SPRED1 and SPRED2 with the novel kinase, DYRK1A. DYRK1A has become the subject of recent research focus as it plays a central role in Caenorhabditis elegans oocyte maturation and egg activation, and there is strong evidence that it could be involved in Down syndrome in humans. Both SPRED1 and SPRED2 inhibit the ability of DYRK1A to phosphorylate its substrates, Tau and STAT3. This inhibition occurs via an interaction of the CRD of the SPREDs with the kinase domain of DYRK1A. DYRK1A substrates must bind to the kinase to enable phosphorylation, and SPRED proteins compete for the same binding site to modify this process. Our accumulated evidence indicates that the SPRED proteins are likely physiological modifiers of DYRK1A.     

Mg2+ in β-TCP/Mg–Zn composite enhances the differentiation of human bone marrow stromal cells into osteoblasts through MAPK-regulated Runx2/Osx

Inducing the osteogenic differentiation from bone marrow stromal cells (BMSCs) might be a potent strategy for treating bone loss and nonunion during fracture and improving fracture healing. Among several signaling pathways involved, mitogen-activated protein kinases (MAPKs) have been reported to play a critical role. Magnesium (Mg)-based alloys, including Mg–Zn alloy, have been used clinically as implants in the musculoskeletal field and could promote BMSC osteogenic differentiation. However, the underlying mechanisms remain unclear. In this study, we produced Mg–Zn alloy consists of Mg and low concentrations of Zn, calcium carbonate, and β-tricalcium phosphate (β-TCP; manifesting process not shown), prepared Mg, Zn, and Mg–Zn extracts, and investigated the specific effects of these extracts on human BMSC (hBMSC) osteogenic differentiation and MAPK signaling. Mg extracts and Mg–Zn extracts could significantly promote the osteogenic differentiation of hBMSCs as manifested as increased alkaline phosphatase levels, enhanced calcium nodules formation, and increased messenger RNA expression and protein levels of osteogenesis markers, including BMPs, Col-I, Runx2, and Osx; in the meantime, Mg culture medium (CM) and Mg–Zn CM both significantly enhanced the activation of MAPK signaling in hBMSCs. By adding ERK1/2 signaling, p38 signaling, or JNK signaling inhibitor to Mg–Zn CM, or conducting p38 MAPK silence in hBMSCs, we revealed that these extracts might promote hBMSC osteogenic differentiation via p38 MAPK signaling and MAPK-regulated Runx2/Osx. In conclusion, Mg²⁺ in β-TCP/Mg–Zn extract promotes the osteogenic differentiation of hBMSCs via MAPK-regulated Runx2/Osx interaction.     

Direct Role for the Replication Protein Treslin (Ticrr) in the ATR Kinase-mediated Checkpoint Response

TopBP1 (topoisomerase IIβ-binding protein 1) is a dual replication/checkpoint protein. Treslin/Ticrr, an essential replication protein, was discovered as a binding partner for TopBP1 and also in a genetic screen for checkpoint regulators in zebrafish. Treslin is phosphorylated by CDK2/cyclin E in a cell cycle-dependent manner, and its phosphorylation state dictates its interaction with TopBP1. The role of Treslin in the initiation of DNA replication has been partially elucidated; however, its role in the checkpoint response remained elusive. In this study, we show that Treslin stimulates ATR phosphorylation of Chk1 both in vitro and in vivo in a TopBP1-dependent manner. Moreover, we show that the phosphorylation state of Treslin at Ser-1000 is important for its checkpoint activity. Overall, our results indicate that, like TopBP1, Treslin is a dual replication/checkpoint protein that directly participates in ATR-mediated checkpoint signaling.     

Extracellular Signal-regulated Kinase Mediates Expression of Arginase II but Not Inducible Nitric-oxide Synthase in Lipopolysaccharide-stimulated Macrophages

The mitogen-activated protein kinases (MAPK) have been shown to participate in iNOS induction following lipopolysaccharide (LPS) stimulation, while the role of MAPKs in the regulation of arginase remains unclear. We hypothesized that different MAPK family members are involved in iNOS and arginase expression following LPS stimulation. LPS-stimulated RAW 264.7 cells exhibited increased protein and mRNA levels for iNOS, arginase I, and arginase II; although the induction of arginase II was more robust than that for arginase I. A p38 inhibitor completely prevented iNOS expression while it only attenuated arginase II induction. In contrast, a MEK1/2 inhibitor (ERK pathway) completely abolished arginase II expression while actually enhancing iNOS induction in LPS-stimulated cells. Arginase II promoter activity was increased by ∼4-fold following LPS-stimulation, which was prevented by the ERK pathway inhibitor. Arginase II promoter activity was unaffected by a p38 inhibitor or JNK pathway interference. Transfection with a construct expressing a constitutively active RAS mutant increased LPS-induced arginase II promoter activity, while transfection with a vector expressing a dominant negative ERK2 mutant or a vector expressing MKP-3 inhibited the arginase II promoter activity. LPS-stimulated nitric oxide (NO) production was increased following siRNA-mediated knockdown of arginase II and decreased when arginase II was overexpressed. Our results demonstrate that while both the ERK and p38 pathways regulate arginase II induction in LPS-stimulated macrophages, iNOS induction by LPS is dependent on p38 activation. These results suggest that differential inhibition of the MAPK pathway may be a potential therapeutic strategy to regulate macrophage phenotype.     

Structural Mechanism for the Specific Assembly and Activation of the Extracellular Signal Regulated Kinase 5 (ERK5) Module

Mitogen-activated protein kinase (MAPK) activation depends on a linear binding motif found in all MAPK kinases (MKK). In addition, the PB1 (Phox and Bem1) domain of MKK5 is required for extracellular signal regulated kinase 5 (ERK5) activation. We present the crystal structure of ERK5 in complex with an MKK5 construct comprised of the PB1 domain and the linear binding motif. We show that ERK5 has distinct protein-protein interaction surfaces compared with ERK2, which is the closest ERK5 paralog. The two MAPKs have characteristically different physiological functions and their distinct protein-protein interaction surface topography enables them to bind different sets of activators and substrates. Structural and biochemical characterization revealed that the MKK5 PB1 domain cooperates with the MAPK binding linear motif to achieve substrate specific binding, and it also enables co-recruitment of the upstream activating enzyme and the downstream substrate into one signaling competent complex. Studies on present day MAPKs and MKKs hint on the way protein kinase networks may evolve. In particular, they suggest how paralogous enzymes with similar catalytic properties could acquire novel signaling roles by merely changing the way they make physical links to other proteins.     

Plasma Kallikrein Promotes Epidermal Growth Factor Receptor Transactivation and Signaling in Vascular Smooth Muscle through Direct Activation of Protease-activated Receptors

The kallikrein-kinin system, along with the interlocking renin-angiotensin system, is a key regulator of vascular contractility and injury response. The principal effectors of the kallikrein-kinin system are plasma and tissue kallikreins, proteases that cleave high molecular weight kininogen to produce bradykinin. Most of the cellular actions of kallikrein (KK) are thought to be mediated by bradykinin, which acts via G protein-coupled B1 and B2 bradykinin receptors on VSMCs and endothelial cells. Here, we find that primary aortic vascular smooth muscle but not endothelial cells possess the ability to activate plasma prekallikrein. Surprisingly, exposing VSMCs to prekallikrein leads to activation of the ERK1/2 mitogen-activated protein kinase cascade via a mechanism that requires kallikrein activity but does not involve bradykinin receptors. In transfected HEK293 cells, we find that plasma kallikrein directly activates G protein-coupled protease-activated receptors (PARs) 1 and 2, which possess consensus kallikrein cleavage sites, but not PAR4. In vascular smooth muscles, KK stimulates ADAM (a disintegrin and metalloprotease) 17 activity via a PAR1/2 receptor-dependent mechanism, leading sequentially to release of the endogenous ADAM17 substrates, amphiregulin and tumor necrosis factor-α, metalloprotease-dependent transactivation of epidermal growth factor receptors, and metalloprotease and epidermal growth factor receptor-dependent ERK1/2 activation. These results suggest a novel mechanism of bradykinin-independent kallikrein action that may contribute to the regulation of vascular responses in pathophysiologic states, such as diabetes mellitus.     

Toll-like Receptor 2 (TLR2), Transforming Growth Factor-β, Hyaluronan (HA), and Receptor for HA-mediated Motility (RHAMM) Are Required for Surfactant Protein A-stimulated Macrophage Chemotaxis

The innate immune system protects the host from bacterial and viral invasion. Surfactant protein A (SPA), a lung-specific collectin, stimulates macrophage chemotaxis. However, the mechanisms regulating this function are unknown. Hyaluronan (HA) and its receptors RHAMM (receptor for HA- mediated motility, CD168) and CD44 also regulate cell migration and inflammation. We therefore examined the role of HA, RHAMM, and CD44 in SPA-stimulated macrophage chemotaxis. Using antibody blockade and murine macrophages, SPA-stimulated macrophage chemotaxis was dependent on TLR2 but not the other SPA receptors examined. Anti-TLR2 blocked SPA-induced production of TGFβ. In turn, TGFβ1-stimulated chemotaxis was inhibited by HA-binding peptide and anti-RHAMM antibody but not anti-TLR2 antibody. Macrophages from TLR2^−/− mice failed to migrate in response to SPA but responded normally to TGFβ1 and HA, effects that were blocked by anti-RHAMM antibody. Macrophages from WT and CD44^−/− mice had similar responses to SPA, whereas those from RHAMM^−/− mice had decreased chemotaxis to SPA, TGFβ1, and HA. In primary macrophages, SPA-stimulated TGFβ production was dependent on TLR2, JNK, and ERK but not p38. Pam3Cys, a specific TLR2 agonist, stimulated phosphorylation of JNK, ERK, and p38, but only JNK and ERK inhibition blocked Pam3Cys-stimulated chemotaxis. We have uncovered a novel pathway for SPA-stimulated macrophage chemotaxis where SPA stimulation via TLR2 drives JNK- and ERK-dependent TGFβ production. TGFβ1, in turn, stimulates macrophage chemotaxis in a RHAMM and HA-dependent manner. These findings are highly relevant to the regulation of innate immune responses by SPA with key roles for specific components of the extracellular matrix.     

A Novel Ste20-related Proline/Alanine-rich Kinase (SPAK)-independent Pathway Involving Calcium-binding Protein 39 (Cab39) and Serine Threonine Kinase with No Lysine Member 4 (WNK4) in the Activation of Na-K-Cl Cotransporters

Na⁺-dependent chloride cotransporters (NKCC1, NKCC2, and NCC) are activated by phosphorylation to play critical roles in diverse physiological responses, including renal salt balance, hearing, epithelial fluid secretion, and volume regulation. Serine threonine kinase WNK4 (With No K = lysine member 4) and members of the Ste20 kinase family, namely SPAK and OSR1 (Ste20-related proline/alanine-rich kinase, Oxidative stress-responsive kinase) govern phosphorylation. According to present understanding, WNK4 phosphorylates key residues within SPAK/OSR1 leading to kinase activation, allowing SPAK/OSR1 to bind to and phosphorylate NKCC1, NKCC2, and NCC. Recently, the calcium-binding protein 39 (Cab39) has emerged as a binding partner and enhancer of SPAK/OSR1 activity, facilitating kinase autoactivation and promoting phosphorylation of the cotransporters. In the present study, we provide evidence showing that Cab39 differentially interacts with WNK4 and SPAK/OSR1 to switch the classic two kinase cascade into a signal kinase transduction mechanism. We found that WNK4 in association with Cab39 activates NKCC1 in a SPAK/OSR1-independent manner. We discovered that WNK4 possesses a domain that bears close resemblance to the SPAK/OSR1 C-terminal CCT/PF2 domain, which is required for physical interaction between the Ste20 kinases and the Na⁺-driven chloride cotransporters. Modeling, yeast two-hybrid, and functional data reveal that this PF2-like domain located downstream of the catalytic domain in WNK4 promotes the direct interaction between the kinase and NKCC1. We conclude that in addition to SPAK and OSR1, WNK4 is able to anchor itself to the N-terminal domain of NKCC1 and to promote cotransporter activation.