Calcium signaling through protein kinases. The Arabidopsis calcium-dependent protein kinase gene family

In plants, numerous Ca(2+)-stimulated protein kinase activities occur through calcium-dependent protein kinases (CDPKs). These novel calcium sensors are likely to be crucial mediators of responses to diverse endogenous and environmental cues. However, the precise biological function(s) of most CDPKs remains elusive. The Arabidopsis genome is predicted to encode 34 different CDPKs. In this Update, we analyze the Arabidopsis CDPK gene family and review the expression, regulation, and possible functions of plant CDPKs. By combining emerging cellular and genomic technologies with genetic and biochemical approaches, the characterization of Arabidopsis CDPKs provides a valuable opportunity to understand the plant calcium-signaling network.     

Cyclic AMP blocks bacterial lipopolysaccharide-induced myosin light chain phosphorylation in endothelial cells through inhibition of Rho/Rho kinase signaling

During Gram-negative sepsis bacterial LPS induces endothelial cell contraction, actin reorganization, and loss of endothelial integrity by an unknown signal mechanism. In this study, we provide evidence that LPS-stimulation of endothelial cells (HUVEC) decreases myosin light chain (MLC) phosphatase, resulting in an increase in MLC phosphorylation followed by cell contraction. All of these LPS effects could be blocked by the Rho-GTPase inhibitor C3 transferase from Clostridium botulinum or the Rho kinase inhibitor Y-27632. These data suggest that LPS induces MLC phosphorylation via Rho/Rho kinase-mediated inhibition of MLC phosphatase in HUVEC. Furthermore, we observed that cAMP-elevating drugs, known to exert a vasoprotective function, mimicked the effects of C3 transferase and Y-27632, i.e., inhibited LPS-induced MLC phosphatase inactivation and MLC phosphorylation. cAMP elevation did not inhibit myosin phosphorylation induced by constitutively active V14Rho or the MLC phosphatase inhibitor calyculin and did not induce phosphorylation of RhoA in HUVEC, indicating inhibition of an upstream regulator of Rho/Rho kinase. Taken together, Rho/Rho kinase appears to be a central target for inflammatory mediators causing endothelial cell contraction such as bacterial toxins, but also for vasoprotective molecules elevating intracellular cAMP.     

p57KIP2 modulates stress-activated signaling by inhibiting c-Jun NH2-terminal kinase/stress-activated protein Kinase

p57KIP2, a member of the Cip/Kip family of enzymes that inhibit several cyclin-dependent kinases, plays a role in many biological events including cell proliferation, differentiation, apoptosis, tumorigenesis and developmental changes. The human p57KIP2 gene is located in chromosome 11p15.5, a region implicated in sporadic cancers and Beckwith-Wiedemann syndrome. We here report that p57KIP2 physically interacts with and inhibits c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK). The carboxyl-terminal QT domain of p57KIP2 is crucial for the inhibition of JNK/SAPK. Overexpressed p57KIP2 also suppressed UV- and MEKK1-induced apoptotic cell death. p57KIP2 expression during C2C12 myoblast differentiation resulted in repression of the JNK activity stimulated by UV light. Furthermore, UV-stimulated JNK1 activity was higher in mouse embryonic fibroblasts derived from p57-/- mice than in the cells from wild-type mice. Taken together, these findings suggest that p57KIP2 modulates stress-activated signaling by functioning as an endogenous inhibitor of JNK/SAPK.     

Modulation of cytosolic calcium signaling by protein kinase A-mediated phosphorylation of inositol 1,4,5-trisphosphate receptors

Calcium release via intracellular Ca2+ release channels is a central event underpinning the generation of numerous, often divergent physiological processes. In electrically non-excitable cells, this Ca2+ release is brought about primarily through activation of inositol 1,4,5-trisphosphate receptors and typically takes the form of calcium oscillations. It is widely believed that information is carried in the temporal and spatial characteristics of these signals. Furthermore, stimulation of individual cells with different agonists can generate Ca2+ oscillations with dramatically different spatial and temporal characteristics. Thus, mechanisms must exist for the acute regulation of Ca2+ release such that agonist-specific Ca2+ signals can be generated. One such mechanism by which Ca2+ signals can be modulated is through simultaneous activation of multiple second messenger pathways. For example, activation of both the InsP3 and cAMP pathways leads to the modulation of Ca2+ release through protein kinase A mediated phosphoregulation of the InsP3R. Indeed, each InsP3R subtype is a potential substrate for PKA, although the functional consequences of this phosphorylation are not clear. This review will focus on recent advances in our understanding of phosphoregulation of InsP3R, as well as the functional consequences of this modulation in terms of eliciting specific cellular events.     

The KDEL receptor modulates the endoplasmic reticulum stress response through mitogen-activated protein kinase signaling cascades

The accumulation of misfolded proteins in the endoplasmic reticulum (ER) evokes the ER stress response. The resultant outcomes are cytoprotective but also proapoptotic. ER chaperones and misfolded proteins exit to the secretory pathway and are retrieved to the ER, during which process the KDEL receptor plays a significant role. Using an expression of a mutant KDEL receptor that lacks the ability for ligand recognition, we show that the impairment of retrieval by the KDEL receptor led to a mis-sorting of the immunoglobulin-binding protein BiP, an ER chaperone that has a retrieval signal from the early secretory pathway, which induced intense ER stress response and an increase in susceptibility to ER stress in HeLa cells. Furthermore, we show that the ER stress response accompanied the activation of p38 mitogen-activated protein (MAP) kinases and c-Jun amino-terminal kinases (JNKs) and that the expression of the mutant KDEL receptor suppressed the activation of p38 and JNK1 but not JNK2. The effect of the expression of the mutant KDEL receptor was consistent with the effect of a specific inhibitor for p38 MAP kinases, because the inhibitor sensitized HeLa cells to ER stress. We also found that activation of the KDEL receptor by the ligand induced the phosphorylation of p38 MAP kinases. These results indicate that the KDEL receptor participates in the ER stress response not only by its retrieval ability but also by modulating MAP kinase signaling, which may affect the outcomes of the mammalian ER stress response.     

Cyclic AMP-specific phosphodiesterase, PDE8A1, is activated by protein kinase A-mediated phosphorylation

The cyclic AMP-specific phosphodiesterase PDE8 has been shown to play a pivotal role in important processes such as steroidogenesis, T cell adhesion, regulation of heart beat and chemotaxis. However, no information exists on how the activity of this enzyme is regulated. We show that under elevated cAMP conditions, PKA acts to phosphorylate PDE8A on serine 359 and this action serves to enhance the activity of the enzyme. This is the first indication that PDE8 activity can be modulated by a kinase, and we propose that this mechanism forms a feedback loop that results in the restoration of basal cAMP levels.     

Cyclic AMP inhibits extracellular signal-regulated kinase and phosphatidylinositol 3-kinase/Akt pathways by inhibiting Rap1

Cyclic AMP inhibited both ERK and Akt activities in rat C6 glioma cells. A constitutively active form of phosphatidylinositol 3-kinase (PI3K) prevented cAMP from inhibiting Akt, suggesting that the inactivation of Akt by cAMP is a consequence of PI3K inhibition. Neither protein kinase A nor Epac (Exchange protein directly activated by cAMP), two known direct effectors of cAMP, mediated the cAMP-induced inhibition of ERK and Akt phosphorylation. Cyclic AMP inhibited Rap1 activation in C6 cells. Moreover, inhibition of Rap1 by a Rap1 GTPase-activating protein-1 also resulted in a decrease in ERK and Akt phosphorylation, which was not further decreased by cAMP, suggesting that cAMP inhibits ERK and Akt by inhibiting Rap1. The role of Rap1 in ERK and Akt activity was further demonstrated by our observation that an active form of Epac, which activated Rap1 in the absence of cAMP, increased ERK and Akt phosphorylation. Inhibition of ERK and/or PI3K pathways mediated the inhibitory effects of cAMP on insulin-like growth factor-I (IGF-I) and IGF-binding protein-3 gene expression. Moreover, cAMP, as well as ERK and PI3K inhibitors produced equivalent stimulation and inhibition, respectively, of p27(Kip1) and cyclin D2 protein levels, potentially explaining the observation that cAMP prevented C6 cells from entering S phase.     

Cyclic GMP modulates the expression of Gi protein and adenylyl cyclase signaling in vascular smooth muscle cells

We have recently shown that the nitric oxide (NO) donor, SNAP, decreased the expression of Giα proteins and associated functions in vascular smooth muscle cells. Because NO stimulates soluble guanylyl cyclase and increases the levels of guanosine 3′,5′-cyclic monophosphate (cGMP), the present studies were undertaken to investigate whether cGMP can also modulate the expression of Gi proteins and associated adenylyl cyclase signaling. A10 vascular smooth muscle cells (VSMCs) and primary cultured cells from aorta of Sprague Dawley rats were used for these studies. The cells were treated with 8-bromoguanosine 3′,5′-cyclic monophosphate (8Br-cGMP) for 24 h and the expression of Giα proteins was determined by immunobloting techniques. Adenylyl cyclase activity was determined by measuring [³²P]cAMP formation for [α-³²P]ATP. Treatment of cells with 8-Br-cGMP (0.5 mM) decreased the expression of Giα-2 and Giα-3 by about 30–45%, which was restored towards control levels by KT5823, an inhibitor of protein kinase G. On the other and hand, the levels of Gsα protein were not altered by this treatment. The decreased expression of Giα proteins by 8Br-cGMP treatment was reflected in decreased Gi functions. For example, the inhibition of forskolin (FSK)-stimulated adenylyl cyclase activity by low concentrations of GTPγS (receptor-independent Gi functions) was significantly decreased by 8Br-cGMP treatment. In addition, exposure of the cells to 8Br-cGMP also resulted in the attenuation of angiotensin (Ang) II- and C-ANP_4–23 (a ring-deleted analog of atrial natriuretic peptide [ANP]-mediated inhibition of adenylyl cyclase activity (receptor-dependent functions of Gi). On the other hand, Gsα-mediated stimulations of adenylyl cyclase by GTPγS, isoproterenol and FSK were significantly augmented in 8Br-cGMP-treated cells. These results indicated the 8Br-cGMP decreased the expression of Giα proteins and associated functions in VSMCs. From these studies, it can be suggested that 8Br-cGMP-induced decreased levels of Gi proteins and resultant increased levels of cAMP may be an additional mechanism through which cGMP regulates vascular tone and thereby blood pressure.     

Numb modulates intestinal epithelial cells toward goblet cell phenotype by inhibiting the Notch signaling pathway

Numb was originally identified as an important cell fate determinant that is asymmetrically inherited during mitosis and controls the fate of sibling cells by inhibiting the Notch signaling pathway in neural tissue. The small intestinal epithelium originates from the division of stem cells that reside in the crypt, which further differentiate into goblet cells, absorptive cells, paneth cells, and enteroendocrine cells. However, Numb's involvement in the differentiation process of intestinal epithelium is largely unknown. In the present study, we confirm that both the Numb mRNA and protein isoforms are expressed in adult mouse intestinal mucosa. Numb protein is ubiquitously expressed throughout the crypt–villus axis of the small intestinal epithelium and is mainly localized to the cytoplasmic membrane. Down-regulation of endogenous Numb using RNA interference in cultured intestinal LS174T cells increased Notch signaling, leading to the up-regulation of Hes1 and the down-regulation of Hath1. Knockdown of Numb alleviated MUC2 protein expression and led to loss of the goblet cell phenotype in LS174Tl cells. Our results provide the first evidence that Numb, an important cell fate determinant, modulates intestinal epithelial cells towards the goblet cell phenotype by inhibiting the Notch signaling pathway.     

Rb protein down-regulates the stress-activated signals through inhibiting c-Jun N-terminal kinase/stress-activated protein kinase

The Rb protein is the product of the retinoblastoma susceptibility gene and loss of Rb function is detected in many types of human cancers. Rb plays important roles in the regulation of cell proliferation, differentiation, senescence, and apoptotic cell death. Here we show that Rb can physically interact with c-Jun NH(2)-terminal kinase/stress-activated protein kinase (JNK/SAPK), thereby inhibiting intracellular signals mediated by JNK/SAPK. Both in vitro binding and in vitro kinase studies suggest that a carboxyl-terminal domain of Rb containing amino acids 768-928 might be crucial for inhibiting JNK/SAPK. In comparison, Rb did not affect enzymatic activity of either extracellular signal-regulated kinase 1 or p38. Ectopically expressed Rb also abrogated the apoptotic cell death induced by ultraviolet radiation or the activation of MEKK1, an upstream kinase that can stimulate the JNK/SAPK cascade. JNK/SAPK inhibition highlights a novel function of Rb, which may provide a new mechanism by which Rb regulates cell death. JNK/SAPK is a major protein kinase that can be stimulated in response to a variety of cellular stresses. Our results, therefore, suggest that Rb, by inhibiting JNK/SAPK, may act as a negative regulator in stress-activated intracellular signaling cascades.     

Differential signaling of cyclic AMP: opposing effects of exchange protein directly activated by cyclic AMP and cAMP-dependent protein kinase on protein kinase B activation.

The recent discovery of Epac, a novel cAMP receptor protein, opens up a new dimension in studying cAMP-mediated cell signaling. It is conceivable that many of the cAMP functions previously attributed to cAMP-dependent protein kinase (PKA) are in fact also Epac-dependent. The finding of an additional intracellular cAMP receptor provides an opportunity to further dissect the divergent roles that cAMP exerts in different cell types. In this study, we probed cross-talk between cAMP signaling and the phosphatidylinositol 3-kinase/PKB pathways. Specifically, we examined the modulatory effects of cAMP on PKB activity by monitoring the specific roles that Epac and PKA play individually in regulating PKB activity. Our study suggests a complex regulatory scheme in which Epac and PKA mediate the opposing effects of cAMP on PKB regulation. Activation of Epac leads to a phosphatidylinositol 3-kinase-dependent PKB activation, while stimulation of PKA inhibits PKB activity. Furthermore, activation of PKB by Epac requires the proper subcellular targeting of Epac. The opposing effects of Epac and PKA on PKB activation provide a potential mechanism for the cell type-specific differential effects of cAMP. It is proposed that the net outcome of cAMP signaling is dependent upon the dynamic abundance and distribution of intracellular Epac and PKA.     

Angiopoietin-like protein 3 modulates barrier properties of human glomerular endothelial cells through a possible signaling pathway involving phosphatidylinositol-3 kinase/protein kinase B and integrin alphaVbeta3

Podocytes can influence glomerular endothelial cell (GEnC) barrier properties and take part in the development of proteinuria by some molecules. Angiopoietin-like protein 3 (Angptl3), secreted by podocytes, is a member of the angiopoietin-like protein family that has important biological functions in endothelial cells. In our previous studies, we showed that mRNA expression of Angptl3 increased significantly in kidneys of children with minimal change nephrotic syndrome. And the mRNA level of Angptl3 was increased in the glomerulus of adriamycin rats with the development of proteinuria. It was also found that Angptl3 was expressed in the cytoplasm of cultured podocytes. Thus, Angptl3 might influence the biological functions of GEnCs in a paracrine manner. In this study, we found that Angptl3 could increase the permeability of GEnCs and increase the level of protein kinase B phosphorylation in cultured GEnCs in vitro. LY294002, a phosphatidylinositol-3 kinase inhibitor, could prevent the increase of permeability of GEnCs induced by Angptl3. Our results also indicated that the integrin αVβ3 antibody (LM609) could block the Angptl3-induced protein kinase B phosphorylation.     

Cell signaling through the protein kinases cAMP-dependent protein kinase, protein kinase Cepsilon, and RAF-1 regulates amphotropic murine leukemia virus envelope protein-induced syncytium formation

Amphotropic murine leukemia virus (A-MuLV) utilizes the PiT2 sodium-dependent phosphate transporter as its cell surface receptor to infect mammalian cells. The process of A-MuLV infection requires cleavage of the R peptide from the envelope protein. This occurs within virions thereby rendering them competent to fuse with target cells. Envelope proteins lacking the inhibitory R peptide (e.g. envelope (R-) proteins) induce viral envelope-mediated cell-cell fusion (syncytium). Here we have performed studies to determine if cell signaling through protein kinases is involved in the regulation of PiT2-mediated A-MuLV envelope (R-)-induced syncytium formation. Truncated A-MuLV retroviral envelope protein lacking the inhibitory R peptide (R-) was used to induce viral envelope-mediated cell-cell fusion. Signaling through cyclic AMP to activate PKA was found to inhibit envelope-induced cell-cell fusion, whereas treatment of cells with PKA inhibitors H89, KT5720, and PKA Catalpha siRNA all enhanced this cell fusion process. It was noted that activation of PKC, as well as overexpression of PKCepsilon, up-regulated A-MuLV envelope protein-induced cell-cell fusion, whereas exposure to PKC inhibitors and expression of a kinase-inactive dominant-negative mutant of PKCepsilon (K437R) inhibited syncytium formation. v-ras transformed NIH3T3 cells were highly susceptible to A-MuLV envelope-induced cell-cell fusion, whereas expression of a dominant-negative mutant of Ras (N17Ras) inhibited this cell fusion process. Importantly, activation of Raf-1 protein kinase also is required for A-MuLV envelope-induced syncytium formation. Expression of constitutively active BXB Raf supported, whereas expression of a dominant-negative mutant of Raf-1 (Raf301) blocked, A-MuLV-induced cell-cell fusion. These results indicate that specific cell signaling components are involved in regulating PiT2-mediated A-MuLV-induced cell-cell fusion. Selective pharmacological modulation of these signaling components may be an effective means of altering cell susceptibility to viral-mediated cytopathic effects.     

Noncanonical notch signaling modulates cytokine responses of dendritic cells to inflammatory stimuli

Dendritic cell (DC)-derived cytokines play a key role in specifying adaptive immune responses tailored to the type of pathogen encountered and the local tissue environment. However, little is known about how DCs perceive the local environment. We investigated whether endogenous Notch signaling could affect DC responses to pathogenic stimuli. We demonstrate that concurrent Notch and TLR stimulation results in a unique cytokine profile in mouse bone-marrow derived DCs characterized by enhanced IL-10 and IL-2, and reduced IL-12 expression compared with TLR ligation alone. Unexpectedly, modulation of cytokine production occurred through a noncanonical Notch signaling pathway, independent of γ-secretase activity. Modulation required de novo protein synthesis, and PI3K, JNK, and ERK activity were necessary for enhanced IL-2 expression, whereas modulation of IL-10 required only PI3K activity. Further, we show that this γ-secretase-independent Notch pathway can induce PI3K activity. In contrast, expression of the canonical Notch target gene Hes1 was suppressed in DCs stimulated with Notch and TLR ligands simultaneously. Thus, our data suggest that Notch acts as an endogenous signal that modulates cytokine expression of DCs through a noncanonical pathway and therefore has the potential to tailor the subsequent adaptive immune response in a tissue- and/or stage-dependent manner.     

Cyclic AMP inhibition of protein kinase activity in HeLa 65 plasma membranes.

Demethylisothiocolchicine forms monoclinic crystals, space group P2₁, a = 11.906, b = 8.713, c = 10.004 Å; β = 89.44°; Z = 2. The crystal and molecular structure was determined from 1911 independent x-ray reflections and refined to R = 0.04. The molecule has the same overall shape as active colchicine derivatives but one methoxy group has a different conformation from that found in other compounds.