Osvaldo Rey

Protein Kinase D Signaling

A rapid increase in the synthesis of lipid-derived second messengers with subsequent activation of protein phosphorylation cascades has emerged as one of the fundamental mechanisms of signal transduction in animal cells. A plethora of external signals, including hormones, neurotransmitters, growth factors, cytokines, bioactive lipids, and tastants promote the stimulation of the isoforms of the PLC family, including , , , and . PLCs catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate to produce two second messengers: inositol 1,4,5-P3, which triggers the release of Ca from internal stores, and DAG, which elicits cellular responses through a variety of effectors (1). The most prominent intracellular targets of DAG are the classic ( , , ) and novel ( , , , ) isoforms of PKC, which are differentially expressed in cells and tissues (2, 3). The mechanisms by which PKC-mediated signals are propagated to critical downstream targets remain incompletely understood. Protein kinase D (PKD), the founding member of a new family of serine/threonine protein kinases and the subject of this minireview, occupies a unique position in the signal transduction pathways initiated by DAG and PKC. PKD not only is a direct DAG target but also lies downstream of PKCs in a novel signal transduction pathway implicated in the regulation of multiple fundamental biological processes.

Amino Acid-stimulated Ca2+ Oscillations Produced by the Ca2+-sensing Receptor Are Mediated by a Phospholipase C/Inositol 1,4,5-Trisphosphate-independent Pathway That Requires G12, Rho, Filamin-A, and the Actin Cytoskeleton

The G protein-coupled Ca2+-sensing receptor (CaR) is an allosteric protein that responds to two different agonists, Ca2+ and aromatic amino acids, with the production of sinusoidal or transient oscillations in intracellular Ca2+ concentration ([Ca2+]i). Here, we examined whether these differing patterns of [Ca2+]i oscillations produced by the CaR are mediated by separate signal transduction pathways. Using real time imaging of changes in phosphatidylinositol 4,5-biphosphate hydrolysis and generation of inositol 1,4,5-trisphosphate in single cells, we found that stimulation of CaR by an increase in the extracellular Ca2+ concentration ([Ca2+]o) leads to periodic synthesis of inositol 1,4,5-trisphosphate, whereas l-phenylalanine stimulation of the CaR does not induce any detectable change in the level this second messenger. Furthermore, we identified a novel pathway that mediates transient [Ca2+]i oscillations produced by the CaR in response to l-phenylalanine, which requires the organization of the actin cytoskeleton and involves the small GTPase Rho, heterotrimeric proteins of the G12 subfamily, the C-terminal region of the CaR, and the scaffolding protein filamin-A. Our model envisages that Ca2+ or amino acids stabilize unique CaR conformations that favor coupling to different G proteins and subsequent activation of distinct downstream signaling pathways.

Protein Kinase Cν/Protein Kinase D3 Nuclear Localization, Catalytic Activation, and Intracellular Redistribution in Response to G Protein-coupled Receptor Agonists

The protein kinase D (PKD) family consists of three serine/threonine kinases: PKCμ/PKD, PKD2, and PKCν/PKD3. Whereas PKD has been the focus of most studies, virtually nothing is known about the effect of G protein-coupled receptor agonists (GPCR) on the regulatory properties and intracellular distribution of PKD3. Consequently, we examined the mechanism that mediates its activation and intracellular distribution. GPCR agonists induced a rapid activation of PKD3 by a protein kinase C (PKC)-dependent pathway that leads to the phosphorylation of the activation loop of PKD3. Comparison of the steady-state distribution of endogenous or tagged PKD3 versus PKD and PKD2 in unstimulated cells indicated that whereas PKD and PKD2 are predominantly cytoplasmic, PKD3 is present both in the nucleus and cytoplasm. This distribution of PKD3 results from its continuous shuttling between both compartments by a mechanism that requires a nuclear import receptor and a competent CRM1-nuclear export pathway. Cell stimulation with the GPCR agonist neurotensin induced a rapid and reversible plasma membrane translocation of PKD3 that is PKC-dependent. Interestingly, the nuclear accumulation of PKD3 can be dramatically enhanced in response to its activation. Thus, this study demonstrates that the intracellular distribution of PKD isoenzymes are distinct, and suggests that their signaling properties are regulated by differential localization.

Sequential Protein Kinase C (PKC)-dependent and PKC-independent Protein Kinase D Catalytic Activation via Gq-coupled Receptors DIFFERENTIAL REGULATION OF ACTIVATION LOOP SER744 AND SER748 PHOSPHORYLATION

Protein kinase D (PKD) is a serine/threonine protein kinase rapidly activated by G protein-coupled receptor (GPCR) agonists via a protein kinase C (PKC)-dependent pathway. Recently, PKD has been implicated in the regulation of long term cellular activities, but little is known about the mechanism(s) of sustained PKD activation. Here, we show that cell treatment with the preferential PKC inhibitors GF 109203X or Gö 6983 blocked rapid (1–5-min) PKD activation induced by bombesin stimulation, but this inhibition was greatly diminished at later times of bombesin stimulation (e.g. 45 min). These results imply that GPCR-induced PKD activation is mediated by early PKC-dependent and late PKC-independent mechanisms. Western blot analysis with site-specific antibodies that detect the phosphorylated state of the activation loop residues Ser744 and Ser748 revealed striking PKC-independent phosphorylation of Ser748 as well as Ser744 phosphorylation that remained predominantly but not completely PKC-dependent at later times of bombesin or vasopressin stimulation (20–90 min). To determine the mechanisms involved, we examined activation loop phosphorylation in a set of PKD mutants, including kinase-deficient, constitutively activated, and PKD forms in which the activation loop residues were substituted for alanine. Our results show that PKC-dependent phosphorylation of the activation loop Ser744 and Ser748 is the primary mechanism involved in early phase PKD activation, whereas PKD autophosphorylation on Ser748 is a major mechanism contributing to the late phase of PKD activation occurring in cells stimulated by GPCR agonists. The present studies identify a novel mechanism induced by GPCR activation that leads to late, PKC-independent PKD activation.

Protein Kinase D Mediates Mitogenic Signaling by Gq-coupled Receptors through Protein Kinase C-independent Regulation of Activation Loop Ser744 and Ser748 Phosphorylation

Rapid protein kinase D (PKD) activation and phosphorylation via protein kinase C (PKC) have been extensively documented in many cell types cells stimulated by multiple stimuli. In contrast, little is known about the role and mechanism(s) of a recently identified sustained phase of PKD activation in response to G protein-coupled receptor agonists. To elucidate the role of biphasic PKD activation, we used Swiss 3T3 cells because PKD expression in these cells potently enhanced duration of ERK activation and DNA synthesis in response to Gq-coupled receptor agonists. Cell treatment with the preferential PKC inhibitors GF109203X or Gö6983 profoundly inhibited PKD activation induced by bombesin stimulation for <15 min but did not prevent PKD catalytic activation induced by bombesin stimulation for longer times (>60 min). The existence of sequential PKC-dependent and PKC-independent PKD activation was demonstrated in 3T3 cells stimulated with various concentrations of bombesin (0.3–10 nm) or with vasopressin, a different Gq-coupled receptor agonist. To gain insight into the mechanisms involved, we determined the phosphorylation state of the activation loop residues Ser744 and Ser748. Transphosphorylation targeted Ser744, whereas autophosphorylation was the predominant mechanism for Ser748 in cells stimulated with Gq-coupled receptor agonists. We next determined which phase of PKD activation is responsible for promoting enhanced ERK activation and DNA synthesis in response to Gq-coupled receptor agonists. We show, for the first time, that the PKC-independent phase of PKD activation mediates prolonged ERK signaling and progression to DNA synthesis in response to bombesin or vasopressin through a pathway that requires epidermal growth factor receptor-tyrosine kinase activity. Thus, our results identify a novel mechanism of Gq-coupled receptor-induced mitogenesis mediated by sustained PKD activation through a PKC-independent pathway.

Negative cross-talk between calcium-sensing receptor and β-catenin signaling systems in colonic epithelium.

Here, we examined the role of the extracellular Ca2+-sensing receptor (CaSR) in the control of colonic epithelial cell proliferation in vivo and changes in β-catenin triggered by CaSR stimulation in human colonic epithelial cells in vitro. The in vivo studies, using a novel Casr intestinal-specific knock-out mouse, indicate that the genetic ablation of the Casr leads to hyperproliferation of colonic epithelial cells, expansion of the proliferative zone, changes in crypt structure, and enhanced β-catenin nuclear localization. The in vitro results indicate that stimulation of the CaSR, by Ca2+ or by the calcimimetic R-568, produced a striking and time-dependent decrease in the phosphorylation of β-catenin at Ser-552 and Ser-675, two amino acid residues that promote β-catenin transcriptional activity. The reduced phosphorylation of β-catenin coincided with a decline in its nuclear localization and a marked redistribution to the plasma membrane. Furthermore, CaSR stimulation promoted a down-regulation of β-catenin-mediated transcriptional activation. These studies demonstrate that signaling pathways emanating from the CaSR control colonic epithelial cell proliferation in vivo and suggest that the mechanism involves regulation of β-catenin phosphorylation.

Extracellular calcium sensing receptor stimulation in human colonic epithelial cells induces intracellular calcium oscillations and proliferation inhibition.

The extracellular Ca2+‐sensing receptor (CaR) is increasingly implicated in the regulation of multiple cellular functions in the gastrointestinal tract, including secretion, proliferation and differentiation of intestinal epithelial cells. However, the signaling mechanisms involved remain poorly defined. Here we examined signaling pathways activated by the CaR, including Ca2+ oscillations, in individual human colon epithelial cells. Single cell imaging of colon‐derived cells expressing the CaR, including SW‐480, HT‐29, and NCM‐460 cells, shows that stimulation of this receptor by addition of aromatic amino acids or by an elevation of the extracellular Ca2+ concentration promoted striking intracellular Ca2+ oscillations. The intracellular calcium oscillations in response to extracellular Ca2+ were of sinusoidal pattern and mediated by the phospholipase C/diacylglycerol/inositol 1,4,5‐trisphosphate pathway as revealed by a biosensor that detects the accumulation of diacylglycerol in the plasma membrane. The intracellular calcium oscillations in response to aromatic amino acids were of transient type, that is, Ca2+ spikes that returned to baseline levels, and required an intact actin cytoskeleton, a functional Rho, Filamin A and the ion channel TRPC1. Further analysis showed that re‐expression and stimulation of the CaR in human epithelial cells derived from normal colon and from colorectal adenocarcinoma inhibits their proliferation. This inhibition was associated with the activation of the signaling pathway that mediates the generation of sinusoidal, but not transient, intracellular Ca2+ oscillations. Thus, these results indicate that the CaR can function in two signaling modes in human colonic epithelial cells offering a potential link between gastrointestinal responses and food/nutrients uptake and metabolism. J. Cell. Physiol. 225: 73–83, 2010.

Requirement of the TRPC1 Cation Channel in the Generation of Transient Ca2+ Oscillations by the Calcium-sensing Receptor

The calcium-sensing receptor (CaR) is an allosteric protein that responds to extracellular Ca2+ ([Ca2+]o) and aromatic amino acids with the production of different patterns of oscillations in intracellular Ca2+ concentration ([Ca2+]i). An increase in [Ca2+]o stimulates phospholipase C-mediated production of inositol 1,4,5-trisphosphate and causes sinusoidal oscillations in [Ca2+]i. Conversely, aromatic amino acid-induced CaR activation does not stimulate phospholipase C but engages an unidentified signaling mechanism that promotes transient oscillations in [Ca2+]i. We show here that the [Ca2+]i oscillations stimulated by aromatic amino acids were selectively abolished by TRPC1 down-regulation using either a pool of small inhibitory RNAs (siRNAs) or two different individual siRNAs that targeted different coding regions of TRPC1. Furthermore, [Ca2+]i oscillations stimulated by aromatic amino acids were also abolished by inhibition of TRPC1 function with an antibody that binds the pore region of the channel. We also show that aromatic amino acid-stimulated [Ca2+]i oscillations can be prevented by protein kinase C (PKC) inhibitors or siRNA-mediated PKCα down-regulation and impaired by either calmodulin antagonists or by the expression of a dominant-negative calmodulin mutant. We propose a model for the generation of CaR-mediated transient [Ca2+]i oscillations that integrates its stimulation by aromatic amino acids with TRPC1 regulation by PKC and calmodulin.

Intracellular redistribution of protein kinase D2 in response to G-protein-coupled receptor agonists ☆

The protein kinase D (PKD) family consists of three serine/threonine protein kinases: PKC mu/PKD, PKD2, and PKC nu/PKD3. While PKD has been the focus of most studies to date, no information is available on the intracellular distribution of PKD2. Consequently, we examined the mechanism that regulates its intracellular distribution in human pancreatic carcinoma Panc-1 cells. Analysis of the intracellular steady-state distribution of fluorescent-tagged PKD2 in unstimulated cells indicated that this kinase is predominantly cytoplasmic. Cell stimulation with the G protein-coupled receptor agonist neurotensin induced a rapid and reversible plasma membrane translocation of PKD2 by a mechanism that requires PKC activity. In contrast to the other PKD isoenzymes, PKD2 activation did not induce its redistribution from the cytoplasm to the nucleus. Thus, this study demonstrates that the regulation of the distribution of PKD2 is distinct from other PKD isoenzymes, and suggests that the differential spatio-temporal localization of these signaling molecules regulates their specific signaling properties.

Protein kinase D2 potentiates MEK/ERK/RSK signaling, c-Fos accumulation and DNA synthesis induced by bombesin in Swiss 3T3 cells.

Protein kinase D (PKD) plays an important role in mediating cellular DNA synthesis in response to G protein‐coupled receptor (GPCR) agonists but the function of other isoforms of the PKD family has been much less explored. Here, we examined whether PKD2 overexpression in Swiss 3T3 cells facilitates DNA synthesis and the activation of the extracellular regulated protein kinase (ERK) pathway in response to the mitogenic GPCR agonist bombesin. We show that PKD2 overexpression markedly potentiated the ability of this agonist to induce DNA synthesis. Addition of bombesin to Swiss 3T3 cells overexpressing PKD2 also induced a striking increase in the duration of MEK/ERK/RSK activation as compared with cultures of control cells. In contrast, neither DNA synthesis nor the duration of ERK activation in response to epidermal growth factor, which acts via protein kinase C/PKD2‐independent pathways, was increased. Furthermore, bombesin promoted a striking accumulation of c‐Fos protein in cells overexpressing PKD2. Our study demonstrates that PKD2, like PKD, facilitates mitogenesis and supports the hypothesis that an increase in the duration of the ERK signaling leading to accumulation of immediate gene products is one of the mechanisms by which isoforms of the PKD family enhance re‐initiation of DNA synthesis by Gq‐coupled receptor activation. J. Cell. Physiol. 211: 781–790, 2007.