Galpha(12) and Galpha(13) interact with Ser/Thr protein phosphatase type 5 and stimulate its phosphatase activity

The Galpha subunits of the G(12) family of heterotrimeric G proteins, defined by Galpha(12) and Galpha(13), are involved in many signaling pathways and diverse cellular functions. In an attempt to elucidate downstream effectors of Galpha(12) for cellular functions, we have performed a yeast two-hybrid screening of a rat brain cDNA library and revealed that Ser/Thr protein phosphatase type 5 (PP5) is a novel effector of Galpha(12) and Galpha(13). PP5 is a newly identified phosphatase and consists of a C-terminal catalytic domain and an N-terminal regulatory tetratricopeptide repeat (TPR) domain [2]. Arachidonic acid was recently shown to activate PP5 phosphatase activity by binding to its TPR domain, however the precise regulatory mechanism of PP5 phosphatase activity is not fully determined. In this study, we show that active forms of Galpha(12) and Galpha(13) specifically interact with PP5 through its TPR domain and activate its phosphatase activity about 2.5-fold. Active forms of Galpha(12) and Galpha(13) also enhance the arachidonic acid-stimulated PP5 phosphatase activity about 2.5-fold. Moreover, we demonstrate that the active form of Galpha(12) translocates PP5 to the cell periphery and colocalizes with PP5. These results propose a new signaling pathway of G(12) family G proteins.     

PI3K和受体型酪氨酸激酶介导家蚕麻痹肽免疫诱导信号的传递

【目的】普遍存在于鳞翅目昆虫中的ENF肽具有非常相似的结构和生理功能, 对昆虫的发育、 免疫、 应激反应等方面都起着重要的调控作用。有研究报道, 作为ENF肽家族成员之一的家蚕Bombyx mori麻痹肽（paralytic peptide, PP）通过激活MAPK来调控家蚕的免疫应答, 但其完整的分子作用机制尚不明确。本研究旨在解析传递家蚕PP免疫诱导信号的分子通路。【方法】首先通过荧光定量PCR检测了多种昆虫细胞系在PP诱导下抗菌肽基因的转录水平, 并利用Western blot检测p38 MAPK的磷酸化水平, 然后利用不同信号传递分子的抑制剂处理BmE细胞筛选参与传递PP免疫诱导信号的分子。【结果】PI3K抑制剂LY294002和受体型酪酸激酶抑制剂Genistein抑制了PP对BmE细胞抗菌肽基因表达的诱导作用; 且BmE细胞和家蚕血细胞在PP的诱导下, Akt和大小约为70 kDa的细胞膜蛋白均出现磷酸化水平的动态变化。【结论】PI3K/Akt信号通路和Genistein敏感性的受体型酪氨酸激酶介导了PP免疫诱导信号的传递。     

Requirement for a hsp90 chaperone-dependent MEK1/2-ERK pathway for B cell antigen receptor-induced cyclin D2 expression in mature B lymphocytes

A requirement for cyclin D2 in G(1)-to-S phase progression has been definitively established in mature B cells stimulated via the B cell antigen receptor (BCR). However, the identity of constituents of the BCR signaling cascade that leads to cyclin D2 accumulation remains incomplete. We report that inhibition of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)-1/2 blocked BCR-induced activation of extracellular signal-regulated kinase (ERK). Inhibition of the MEK1/2-ERK pathway was sufficient to abrogate BCR-induced cyclin D2 expression at the mRNA and protein levels. Disruption of endogenous heat shock protein 90 (hsp90) function with geldanamycin abrogated BCR-induced cyclin D2 expression and proliferation. Geldanamycin effects were attributed to a selective depletion of cellular Raf-1 that interrupted BCR-coupled activation of MEK1/2 and ERK. By contrast, signaling through the phosphatidylinositol 3-kinase and protein kinase C pathways was not affected, suggesting that disruption of hsp90 function did not cause a general impairment of BCR signaling. These results suggest that the MEK1/2-ERK pathway is essential for BCR signaling to cyclin D2 accumulation in ex vivo splenic B lymphocytes. Furthermore, these findings imply that hsp90 function is required for BCR signaling through the Raf-1-MEK1/2-ERK pathway but not through the phosphatidylinositol 3-kinase- or protein kinase C-dependent pathways.     

Novel signaling pathways contributing to vascular changes in hypertension

In hypertension, increased peripheral resistance maintains elevated levels of arterial blood pressure. The increase in peripheral resistance results, in part, from abnormal constrictor and dilator responses and vascular remodeling. In this review, we consider four cellular signaling pathways as possible explanations for these abnormal vascular responses: (1) augmented signaling via the epidermal growth factor receptor to cause remodeling of the cerebrovasculature; (2) reduced sphingolipid signaling leading to blunted vasodilation and increased smooth muscle proliferation; (3) increased signaling via Rho/Rho kinase leading to enhanced vasoconstriction, and (4) a relative state of microtubular depolymerization favoring vasoconstriction in hypertension. These novel cell signaling pathways provide new pharmacological targets to reduce total peripheral vascular resistance in hypertension.     

Small G Proteins Rac1 and Ras Regulate Serine/Threonine Protein Phosphatase 5 (PP5)·Extracellular Signal-Regulated Kinase (ERK) Complexes Involved in the Feedback Regulation of Raf1

Serine/threonine protein phosphatase 5 (PP5, PPP5C) is known to interact with the chaperonin heat shock protein 90 (HSP90) and is involved in the regulation of multiple cellular signaling cascades that control diverse cellular processes, such as cell growth, differentiation, proliferation, motility, and apoptosis. Here, we identify PP5 in stable complexes with extracellular signal-regulated kinases (ERKs). Studies using mutant proteins reveal that the formation of PP5·ERK1 and PP5·ERK2 complexes partially depends on HSP90 binding to PP5 but does not require PP5 or ERK1/2 activity. However, PP5 and ERK activity regulates the phosphorylation state of Raf1 kinase, an upstream activator of ERK signaling. Whereas expression of constitutively active Rac1 promotes the assembly of PP5·ERK1/2 complexes, acute activation of ERK1/2 fails to influence the phosphatase-kinase interaction. Introduction of oncogenic HRas (HRas^V12) has no effect on PP5-ERK1 binding but selectively decreases the interaction of PP5 with ERK2, in a manner that is independent of PP5 and MAPK/ERK kinase (MEK) activity, yet paradoxically requires ERK2 activity. Additional studies conducted with oncogenic variants of KRas4B reveal that KRas^L61, but not KRas^V12, also decreases the PP5-ERK2 interaction. The expression of wild type HRas or KRas proteins fails to reduce PP5-ERK2 binding, indicating that the effect is specific to HRas^V12 and KRas^L61 gain-of-function mutations. These findings reveal a novel, differential responsiveness of PP5-ERK1 and PP5-ERK2 interactions to select oncogenic Ras variants and also support a role for PP5·ERK complexes in regulating the feedback phosphorylation of PP5-associated Raf1.     

HGF/SF-met signaling in the control of branching morphogenesis and invasion

Hepatocyte growth factor/Scatter factor (HGF/SF) is a multifunctional growth factor which can induce diverse biological events. In vitro, these include scattering, invasion, proliferation and branching morphogenesis. In vivo, HGF/SF is responsible for many processes during embryonic development and a variety of activities in adults, and many of these normal activities have been implicated in its role in tumorgenesis and metastasis. The c-Met receptor tyrosine kinase is the only known receptor for HGF/SF and mediates all HGF/SF induced biological activities. Upon HGF/SF stimulation, the c-Met receptor is tyrosine-phosphorylated which is followed by the recruitment of a group of signaling molecules and/or adaptor proteins to its cytoplasmic domain and its multiple docking sites. This action leads to the activation of several different signaling cascades that form a complete network of intra and extracellular responses. Different combinations of signaling pathways and signaling molecules and/or differences in magnitude of responses contribute to these diverse series of HGF/SF-Met induced activities and most certainly are influenced by cell type as well as different cellular environments. In this review, we focus on HGF/SF-induced branching morphogenesis and invasion, and bring together recent new findings which provide insight into how HGF/SF, via c-Met induces this response.     

Extracellular matrix-mediated signaling by dentin phosphophoryn involves activation of the Smad pathway independent of bone morphogenetic protein

Cells have ingenious mechanisms for interpreting complex signals from their external microenvironment. Previously, we have shown that phosphophoryn (PP) regulates the expression of bone/dentin marker genes via the integrin/MAPK signaling pathway (Jadlowiec, J., Koch, H., Zhang, X., Campbell, P. G., Seyedain, M., and Sfeir, C. (2004) J. Biol. Chem. 279, 53323-53330). We hypothesize that other signaling pathways important for mineralized tissue morphogenesis such as the Smad pathway could be involved in PP signaling. We determined activation of the Smad pathway in human adult mesenchymal stem cells following treatment with recombinant PP (rPP). We observed that PP enhanced phosphorylation of Smad1 within 30 min and Smad1 translocation to the nucleus within 1 h. PP up-regulated the expression of Smad1 target genes, Smad6, Dlx5, and Runx2. The timing of PP activation of Smad1 implies this is a direct effect; however, we also investigated the possible involvement of bone morphogenetic proteins in PP stimulation of the Smad pathway. PP was shown to up-regulate Bmp-2 gene expression 12 h post-treatment with PP, which is much later than initial detection of Smad1 phosphorylation at 30 min. Furthermore, addition of Noggin did not block Smad1 phosphorylation by PP. We propose that PP could signal via the Smad pathway by either directly stimulating the phosphorylation of Smad1 via integrins or other mechanisms. These might include integrin/bone morphogenetic protein receptor interactions or involvement of PP with other growth factors leading to the modulation of intracellular signaling. It is noteworthy that a non-transforming growth factor-beta family member activates the Smad pathway. The role of PP in regulating the Smad pathway raises very interesting questions regarding the role of PP during bone and tooth development.     

Protein phosphatase 2A: the Trojan Horse of cellular signaling

Dynamic phosphorylation and dephosphorylation of proteins are fundamental mechanisms utilized by cells to transduce signals. Whereas transduction by protein kinases has been a major focus of studies in the last decade, protein phosphatase 2A (PP2A) enzymes emerge in this millenium as the most fashionable players in cellular signaling. Viral proteins target specific PP2A enzymes in order to deregulate chosen cellular pathways in the host and promote viral progeny. The observation that a variety of viruses utilize PP2A to alienate cellular behavior emphasizes the fundamental importance of PP2A in signal transduction. This review will primarily focus on discussing the uniqueness of PP2A regulation and uncovering the critical role played by protein-protein interactions in the modulation of PP2A signaling. Moreover, the place of PP2A in signaling pathways and its functional significance for human diseases will be discussed.     

Protein kinase A and two phosphatases are components of the inositol 1,4,5-trisphosphate receptor macromolecular signaling complex

The inositol 1,4,5-trisphosphate receptor (IP3R) is a ubiquitously expressed intracellular calcium (Ca(2+)) release channel on the endoplasmic reticulum. IP3Rs play key roles in controlling Ca(2+) signals that activate numerous cellular functions including T cell activation, neurotransmitter release, oocyte fertilization and apoptosis. There are three forms of IP3R, all of which are ligand-gated channels activated by the second messenger inositol 1,4,5-trisphosphate. Channel function is modulated via cross-talk with other signaling pathways including those mediated by kinases and phosphatases. In particular IP3Rs are known to be regulated by cAMP-dependent protein kinase (PKA) phosphorylation. In the present study we show that PKA and the protein phosphatases PP1 and PP2A are components of the IP3R1 macromolecular signaling complex. PKA phosphorylation of IP3R1 increases channel activity in planar lipid bilayers. These studies indicate that regulation of IP3R1 function via PKA phosphorylation involves components of a macromolecular signaling complex.