Effects of In Vitro Interactions of Oviduct Epithelial Cells with Frozen-Thawed Stallion Spermatozoa on Their Motility, Viability and Capacitation Status.

Cryopreservation by negatively affecting sperm quality decreases the efficiency of assisted reproduction techniques (ARTs). Thus, we first evaluated sperm motility at different conditions for the manipulation of equine cryopreserved spermatozoa. Higher motility was observed when spermatozoa were incubated for 30 min at 30 × 106/mL compared to lower concentrations (p < 0.05) and when a short centrifugation at 200× g was performed (p < 0.05). Moreover, because sperm suitable for oocyte fertilization is released from oviduct epithelial cells (OECs), in response to the capacitation process, we established an in vitro OEC culture model to select a sperm population with potential fertilizing capacity in this species. We demonstrated E-cadherin and cytokeratin expression in cultures of OECs obtained. When sperm-OEC cocultures were performed, the attached spermatozoa were motile and presented an intact acrosome, suggesting a selection by the oviductal model. When co-cultures were incubated in capacitating conditions a greater number of alive (p < 0.05), capacitated (p < 0.05), with progressive motility (p < 0.05) and with the intact acrosome sperm population was observed (p < 0.05) suggesting that the sperm population released from OECs in vitro presents potential fertilizing capacity. Improvements in handling and selection of cryopreserved sperm would improve efficiencies in ARTs allowing the use of a population of higher-quality sperm.

Metformin inhibition of colorectal cancer cell migration is associated with rebuilt adherens junctions and FAK downregulation.

E-cadherin, a central component of the adherens junction (AJ), is a single-pass transmembrane protein that mediates cell-cell adhesion. The loss of E-cadherin surface expression, and therefore cell-cell adhesion, leads to increased cell migration and invasion. Treatment of colorectal cancer (CRC)-derived cells (SW-480 and HT-29) with 2.0 mM metformin promoted a redistribution of cytosolic E-cadherin to de novo formed puncta along the length of the contacting membranes of these cells. Metformin also promoted translocation from the cytosol to the plasma membrane of p120-catenin, another core component of the AJs. Furthermore, E-cadherin and p120-catenin colocalized with ß-catenin at cell-cell contacts. Western blot analysis of lysates of CRC-derived cells revealed a substantial metformin-induced increase in the level of p120-catenin as well as E-cadherin phosphorylation on Ser838/840 , a modification associated with ß-catenin/E-cadherin interaction. These modifications in E-cadherin, p120-catenin and ß-catenin localization suggest that metformin induces rebuilding of AJs in CRC-derived cells. Those modifications were accompanied by the inhibition of focal adhesion kinase (FAK), as revealed by a significant decrease in the phosphorylation of FAK at Tyr397 and paxillin at Tyr118 . These changes were associated with a reduction in the numbers, but an increase in the size, of focal adhesions and by the inhibition of cell migration. Overall, these observations indicate that metformin targets multiple pathways associated with CRC development and progression.

p.L571P in the linker domain of rat thyroglobulin causes intracellular retention.

Thyroglobulin (TG), a large glycosylated protein secreted by thyrocytes into the thyroid follicular lumen, plays an essential role in thyroid hormone biosynthesis. Rattus norvegicus TG (rTG) is encoded by a large single copy gene, 186-kb long, located on chromosome 7 composed of 48 exons encoding a 8461-kb mRNA. Although the TG gene displays sequence variability, many missense mutations do not impose any adverse effect on the TG protein, whereas other nucleotide substitutions may affect its TG stability and/or TG intracellular trafficking. In order to gain a further understanding of the protein domains regulating its intracellular fate, we cloned a full-length cDNA from rTG into the pcDNA6/V5-His B expression vector. However, transient expression of the cDNA in HEK293T cells showed that the encoded protein was not a wild-type molecule, as it was unable to be secreted in the culture supernatant. Sequencing analyses revealed three random mutations, which accidentally emerged during the course of cloning: c.1712T>C [p.L571P] in the linker domain (amino acid positions 360 to 604), c.2027A>G [p.Q676R] in TG type 1-6 repeat and c.2720A>G [p.Q907R] in the TG type 1-7 repeat. Expression of cDNAs encoding a combination of two mutations [p.Q676R-p.Q907R], [p.L571P-p.Q907R] or [p.L571P-p.Q676R] indicated that any TG bearing the p.L571P substitution was trapped intracellularly. Indeed, we expressed the single point mutant p.L571P and confirmed that this point mutation was sufficient to cause intracellular retention of mutant TG in HEK293T cells. Endo H analysis showed that the p.L571P mutant is completely sensitive to the enzyme, whereas the will-type TG acquires full N-glycan modifications in Golgi apparatus. This data suggest that the p.L571P mutant contains the mannose-type N-glycan, that was added at the first stage of glycosylation. Complex-type N-glycan formation in the Golgi apparatus does not occur, consistent with defective endoplasmic reticulum exit of the mutant TG. Moreover, predictive analysis of the 3D linker domain showed that the p.L571P mutation would result in a significant protein conformational change. In conclusion, our studies identified a novel amino acid residue within the linker domain of TG associated with its conformational maturation and intracellular trafficking.

Metformin inhibits ß-catenin phosphorylation on Ser-552 through an AMPK/PI3K/Akt pathway in colorectal cancer cells.

Several epidemiologic studies have revealed strong inverse associations between metformin use and risk of colorectal cancer development. Nevertheless, the underlying mechanisms are still uncertain. The Wnt/ß-catenin pathway, which plays a central role in intestinal homeostasis and sporadic colorectal cancer development, is regulated by phosphorylation cascades that are dependent and independent of Wnt. Here we report that a non-canonical Ser552 phosphorylation in ß-catenin, which promotes its nuclear accumulation and transcriptional activity, is blocked by metformin via AMPK-mediated PI3K/Akt signaling inhibition.

Protein kinase D1 inhibition interferes with mitosis progression.

Protein kinase D1 (PKD1) plays a vital role in signal transduction, cell proliferation, membrane trafficking, and cancer; however, the majority of the studies up to date had centered primarily on PKD1 functions in interphase, very little is known about its role during cell division. We previously demonstrated that during mitosis PKD1 is activated and associated with centrosomes, spindles, and midbodies. However, these observations did not address whether PKD1 was associated with mitosis regulation. Accordingly, we used rapidly acting PKD-specific inhibitors to examine the contribution of PKD1 the sequence of events in mitosis. We found that although PKD1 overexpression did not affect mitosis progression, suppression of its catalytic activity by two structurally unrelated inhibitors (kb NB 142-70 and CRT 0066101) induced a significant delay in metaphase to anaphase transition time. PKD1 inhibition during mitosis also produced the appearance of abnormal spindles, defects in chromosome alignment, and segregation as well as apoptosis. Thus, these observations indicate that PKD1 activity is associated with mitosis regulation.

Lysosomal permeabilization and endoplasmic reticulum stress mediate the apoptotic response induced after photoactivation of a lipophilic zinc(II) phthalocyanine.

We have previously reported that the phototoxic action of the lipophilic phthalocyanine Pc9 (2,9(10),16(17),23(24) tetrakis[(2-dimethylamino)ethylsulfanyl]phthalocyaninatozinc(II)) encapsulated into poloxamine micelles is related to the induction of an apoptotic response in murine colon CT26 carcinoma cells. In the present study, we explored the intracellular signals contributing to the resulting apoptotic death. We found that Pc9-T1107 arrests cell cycle progression immediately after irradiation promoting then an apoptotic response. Thus, 3 h after irradiation the percentage of hypodiploid cells increased from 5.9 ±â€¯0.6% to 23.1 ±â€¯0.1%; activation of caspases 8 and 9 was evident; the population of cells with loss of mitochondrial membrane potential increased from 1.1 ±â€¯0.4% to 44.0 ±â€¯9.3%; the full-length forms of Bid and PARP-1 were cleaved; and a 50% decrease of the expression levels of the anti-apoptotic proteins Bcl-2 and Bcl-XL was detected. We also found that the photosensitizer, mainly retained in lysosomes and endoplasmic reticulum (ER), promotes the permeabilization of lysosomal membranes and induces ER stress. Lysosomal membrane permeabilization was demonstrated by the reduction of acridine orange lysosome fluorescence, the release of Cathepsin D into the cytosol and ∼50% decrease of Hsp70, a chaperone recognized as a lysosomal stabilizer. Cathepsin D also contributed to Bid cleavage and caspase 8 activation. The oxidative damage to the ER induced an unfolded protein response characterized, 3 h after irradiation, by a 3-fold increase in cytosolic Ca2+ levels and 3-4 times higher expression of ER chaperones GRP78/BIP, calnexin, Hsp90 and Hsp110. The cell death signaling promoted by cytosolic Ca2+, calpains and lysosomal proteases was partially abolished by the Ca2+ chelator BAPTA-AM, the calpain inhibitor PD 150606 and proteases inhibitors. Furthermore, Bax down-regulation observed in Pc9-treated cells was undetectable in the presence of PD 150606, indicating that calpains contribute to Bax proteolytic damage. In summary, our results indicate that photoactivation of Pc9-T1107 led to lysosomal membrane permeabilization, induction of ER stress and activation of a caspase-dependent apoptotic cell death.

Protein kinase D1 (PKD1) phosphorylation on Ser203 by type I p21-activated kinase (PAK) regulates PKD1 localization.

Although PKC-mediated phosphorylation of protein kinase D1 (PKD1) has been extensively characterized, little is known about PKD1 regulation by other upstream kinases. Here we report that stimulation of epithelial or fibroblastic cells with G protein-coupled receptor agonists, including angiotensin II or bombesin, induced rapid and persistent PKD1 phosphorylation at Ser203, a highly conserved residue located within the PKD1 N-terminal domain. Exposure to PKD or PKC family inhibitors did not prevent PKD1 phosphorylation at Ser203, indicating that it is not mediated by autophosphorylation. In contrast, several lines of evidence indicated that the phosphorylation of PKD1 at Ser203 is mediated by kinases of the class I PAK subfamily, specifically 1) exposing cells to four structurally unrelated PAK inhibitors (PF-3758309, FRAX486, FRAX597, and IPA-3) that act via different mechanisms abrogated PKD1 phosphorylation at Ser203, 2) siRNA-mediated knockdown of PAK1 and PAK2 in IEC-18 and Swiss 3T3 cells blunted PKD1 phosphorylation at Ser203, 3) phosphorylation of Ser203 markedly increased in vitro when recombinant PKD1 was incubated with either PAK1 or PAK2 in the presence of ATP. PAK inhibitors did not interfere with G protein-coupled receptor activation-induced rapid translocation of PKD1 to the plasma membrane but strikingly prevented the dissociation of PKD1 from the plasma membrane and blunted the phosphorylation of nuclear targets, including class IIa histone deacetylases. We conclude that PAK-mediated phosphorylation of PKD1 at Ser203 triggers its membrane dissociation and subsequent entry into the nucleus, thereby regulating the phosphorylation of PKD1 nuclear targets, including class IIa histone deacetylases.

Intracellular Ca(2+) oscillations generated via the extracellular Ca(2+)-sensing receptor (CaSR) in response to extracellular Ca(2+) or L-phenylalanine: Impact of the highly conservative mutation Ser170Thr.

The extracellular Ca(2+)-sensing receptor (CaSR) is an allosteric protein that responds to changes in the extracellular concentration of Ca(2+) ([Ca(2+)]e) and aromatic amino acids with the production of different patterns of oscillations in intracellular Ca(2+) concentration ([Ca(2+)]i). An increase in [Ca(2+)]e stimulates sinusoidal oscillations in [Ca(2+)]i whereas aromatic amino acid-induced CaR activation in the presence of a threshold [Ca(2+)]e promotes transient oscillations in [Ca(2+)]i. Here, we examined spontaneous and ligand-evoked [Ca(2+)]i oscillations in single HEK-293 cells transfected with the wild type CaSR or with a mutant CaSR in which Ser170 was converted to Thr (CaSRS170T). Our analysis demonstrates that cells expressing CaSRS170T display [Ca(2+)]i oscillations in the presence of low concentrations of extracellular Ca(2+) and respond to L-Phe with robust transient [Ca(2+)]i oscillations. Our results indicate that the S170T mutation induces a marked increase in CaSR sensitivity to [Ca(2+)]e and imply that the allosteric regulation of the CaSR by aromatic amino acids is not only mediated by an heterotropic positive effect on Ca(2+) binding cooperativity but, as biased agonists, aromatic amino acids stabilize a CaSR conformation that couples to a different signaling pathway leading to transient [Ca(2+)]i oscillations.

Intracellular Ca2+ oscillations generated via the Ca2+-sensing receptor are mediated by negative feedback by PKCα at Thr888.

To clarify the mechanism(s) underlying intracellular Ca(2+) concentration ([Ca(2+)]i) oscillations induced by an elevation in extracellular Ca(2+) concentration ([Ca(2+)]e) via the extracellular Ca(2+)-sensing receptor (CaR), we analyzed the pattern of [Ca(2+)]i response in multiple (2,303) individual HEK-293 cells transfected with the human CaR. An increase in the [Ca(2+)]e from 1.5 to 3 mM produced oscillatory fluctuations in [Ca(2+)]i in 70% of the cell population. To determine the role of PKC in the generation of [Ca(2+)]i oscillations, cells were exposed to increasing concentrations (0.5-5 µM) of the preferential PKC inhibitor Ro-31-8220 before stimulation by extracellular Ca(2+). Ro-31-8220 at 3-5 µM completely eliminated the [Ca(2+)]e-evoked [Ca(2+)]i oscillations and transformed the pattern to a peak and sustained plateau response. Treatment with other broad PKC inhibitors, including GFI or Gö6983, produced an identical response. Similarly, treatment with Ro-31-8220 or GFI eliminated [Ca(2+)]e-evoked [Ca(2+)]i oscillations in colon-derived SW-480 cells expressing the CaR. Treatment with inhibitors targeting classic PKCs, including Gö6976 and Ro-32-0432 as well as small interfering RNA-mediated knockdown of PKCα, strikingly reduced the proportion of cell displaying [Ca(2+)]e-evoked [Ca(2+)]i oscillations. Furthermore, none of the cells analyzed expressing a CaR mutant in which the major PKC phosphorylation site Thr(888) was converted to alanine (CaRT888A) showed [Ca(2+)]i oscillations after CaR activation. Our results show that [Ca(2+)]i oscillations induced by activation of the CaR in response to an increase in extracellular Ca(2+) or exposure to the calcimimetic R-568 result from negative feedback involving PKCα-mediated phosphorylation of the CaR at Thr(888).

Nerve injury induces glial cell line-derived neurotrophic factor (GDNF) expression in Schwann cells through purinergic signaling and the PKC-PKD pathway.

Upon peripheral nerve injury, specific molecular events, including increases in the expression of selected neurotrophic factors, are initiated to prepare the tissue for regeneration. However, the mechanisms underlying these events and the nature of the cells involved are poorly understood. We used the injury-induced upregulation of glial cell-derived neurotrophic factor (GDNF) expression as a tool to gain insights into these processes. We found that both myelinating and nonmyelinating Schwann cells are responsible for the dramatic increase in GDNF expression after injury. We also demonstrate that the GDNF upregulation is mediated by a signaling cascade involving activation of Schwann cell purinergic receptors, followed by protein kinase C signaling which activates protein kinase D (PKD), which leads to increased GDNF transcription. Given the potent effects of GDNF on survival and repair of injured peripheral neurons, we propose that targeting these pathways may yield therapeutic tools to treat peripheral nerve injury and neuropathies.

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

Here, we examined the role of the extracellular Ca(2+)-sensing receptor (CaSR) in the control of colonic epithelial cell proliferation in vivo and changes in ß-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 ß-catenin nuclear localization. The in vitro results indicate that stimulation of the CaSR, by Ca(2+) or by the calcimimetic R-568, produced a striking and time-dependent decrease in the phosphorylation of ß-catenin at Ser-552 and Ser-675, two amino acid residues that promote ß-catenin transcriptional activity. The reduced phosphorylation of ß-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 ß-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 ß-catenin phosphorylation.

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

The extracellular Ca(2+)-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 Ca(2+) 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 Ca(2+) concentration promoted striking intracellular Ca(2+) oscillations. The intracellular calcium oscillations in response to extracellular Ca(2+) 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, Ca(2+) 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 Ca(2+) 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.

Amino acid sensing by enteroendocrine STC-1 cells: role of the Na+-coupled neutral amino acid transporter 2.

The results presented here show that STC-1 cells, a model of intestinal endocrine cells, respond to a broad range of amino acids, including l-proline, l-serine, l-alanine, l-methionine, l-glycine, l-histidine, and alpha-methyl-amino-isobutyric acid (MeAIB) with a rapid increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)). We sought to identify the mechanism by which amino acids induce Ca(2+) signaling in these cells. Several lines of evidence suggest that amino acid transport through the Na(+)-coupled neutral amino acid transporter 2 (SNAT2) is a major mechanism by which amino acids induced Ca(2+) signaling in STC-1 cells: 1) the amino acid efficacy profile for inducing Ca(2+) signaling in STC-1 cells closely matches the amino acid specificity of SNAT2; 2) amino acid-induced Ca(2+) signaling in STC-1 cells was suppressed by removing Na(+) from the medium; 3) the nonmetabolized synthetic substrate of amino acid transport MeAIB produced a marked increase in [Ca(2+)](i); 4) transfection of small interfering RNA targeting SNAT2 produced a marked decrease in Ca(2+) signaling in response to l-proline in STC-1 cells; 5) amino acid-induced increase in [Ca(2+)](i) was associated with membrane depolarization and mediated by Ca(2+) influx, since it depended on extracellular Ca(2+); 6) the increase in [Ca(2+)](i) in response to l-proline, l-alanine, or MeAIB was abrogated by either nifedipine (1-10 muM) or nitrendipine (1 muM), which block L-type voltage-sensitive Ca(2+) channels. We hypothesize that the inward current of Na(+) associated with the function of SNAT2 leads to membrane depolarization and activation of voltage-sensitive Ca(2+) channels that mediate Ca(2+) influx, thereby leading to an increase in the [Ca(2+)](i) in enteroendocrine STC-1 cells.

CID755673 enhances mitogenic signaling by phorbol esters, bombesin and EGF through a protein kinase D-independent pathway.

Recently, CID755673 was reported to act as a highly selective inhibitor of protein kinase D (PKD). In the course of experiments using CID755673, we noticed that it exerted unexpected stimulatory effects on [(3)H]thymidine incorporation and cell cycle progression in Swiss 3T3 cells stimulated by bombesin, a Gq-coupled receptor agonist, phorbol 12,13-dibutyrate (PDBu), a biologically active tumor promoting phorbol ester and epidermal growth factor (EGF). These stimulatory effects could be dissociated from the inhibitory effect of CID755673 on PKD activity, since enhancement of DNA synthesis was still evident in cells with severely down-regulated PKD1 after transfection of siRNA targeting PKD1. A major point raised by our study is that CID755673 can not be considered a specific inhibitor of PKD and it should be used with great caution in experiments attempting to elucidate the role of PKD family members in cellular regulation, particularly cell cycle progression from G(1)/G(o) to S phase.

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 G(q)-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 G(q)-coupled receptor agonist. To gain insight into the mechanisms involved, we determined the phosphorylation state of the activation loop residues Ser(744) and Ser(748). Transphosphorylation targeted Ser(744), whereas autophosphorylation was the predominant mechanism for Ser(748) in cells stimulated with G(q)-coupled receptor agonists. We next determined which phase of PKD activation is responsible for promoting enhanced ERK activation and DNA synthesis in response to G(q)-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 G(q)-coupled receptor-induced mitogenesis mediated by sustained PKD activation through a PKC-independent pathway.

Protein kinase D isozymes activation and localization during mitosis.

The protein kinase D (PKD) family consists of three serine/threonine protein kinases involved in the regulation of fundamental biological processes in response to their activation and intracellular redistribution. Although a substantial amount of information is available describing the mechanisms regulating the activation and intracellular distribution of the PKD isozymes during interphase, nothing is known of their activation status, localization and role during mitosis. The results presented in this study indicate that during mitosis, PKD3 and PKD are phosphorylated at Ser(731) and Ser(744) within their activation loop by a mechanism that requires protein kinase C. Mitosis-associated PKD3 Ser(731) and PKD Ser(744) phosphorylation is related to the catalytic activation of these kinases as evidenced by in vivo phosphorylation of histone deacetylase 5, a substrate of PKD and PKD3. Activation loop-phosphorylated PKD3 and PKD, as well as PKD2, associate with centrosomes, spindles and midbody suggesting that these activated kinases establish dynamic interactions with the mitotic apparatus. Thus, this study reveals a connection between the PKD isozymes and cell division, suggesting a novel role for this family of serine/threonine kinases.

Sequential protein kinase C (PKC)-dependent and PKC-independent protein kinase D catalytic activation via Gq-coupled receptors: differential regulation of activation loop Ser(744) and Ser(748) 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 Ser(744) and Ser(748) revealed striking PKC-independent phosphorylation of Ser(748) as well as Ser(744) 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 Ser(744) and Ser(748) is the primary mechanism involved in early phase PKD activation, whereas PKD autophosphorylation on Ser(748) 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.

Insulin potentiates Ca2+ signaling and phosphatidylinositol 4,5-bisphosphate hydrolysis induced by Gq protein-coupled receptor agonists through an mTOR-dependent pathway.

Multiple lines of evidence support the existence of crosstalk between the insulin receptor and G protein-coupled receptor (GPCR) signaling systems. However, the precise molecular mechanism(s) mediating this interaction is poorly understood. The results presented in this study show that exposure of ductal pancreatic adenocarcinoma BxPc-3, HPAF-II, and PANC-1 cells to insulin for as little as 1 min rapidly enhanced the magnitude and the rate of increase in intracellular Ca2+ concentration produced by the GPCR agonists bradykinin, angiotensin II, vasopressin, neurotensin, and bombesin. The potentiating effect of insulin was dose dependent, and it was produced in response to Gq protein-coupled, but not Gi protein-coupled, receptor agonists. Real-time imaging of single cells showed that treatment with insulin enhances the rate and magnitude of phosphatidylinositol 4,5-bisphosphate hydrolysis and generation of inositol 1,4,5-trisphosphate in response to GPCR stimulation. Short-term treatment with rapamycin, an mTOR (mammalian target of rapamycin) inhibitor, completely abrogated the ability of insulin to increase the rate and magnitude of Ca2+ signaling and production of inositol 1,4,5-trisphosphate in response to bradykinin stimulation, indicating that insulin potentiates Gq protein-coupled receptor signaling through an mTOR-dependent pathway. We propose that the potentiation of GPCR signaling by insulin provides a mechanism by which insulin enhances cellular responsiveness to Gq protein-coupled receptor agonists, including GPCR-mediated autocrine and paracrine loops in cancer cells.

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.

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 Ca(2+) ([Ca(2+)](o)) and aromatic amino acids with the production of different patterns of oscillations in intracellular Ca(2+) concentration ([Ca(2+)](i)). An increase in [Ca(2+)](o) stimulates phospholipase C-mediated production of inositol 1,4,5-trisphosphate and causes sinusoidal oscillations in [Ca(2+)](i). Conversely, aromatic amino acid-induced CaR activation does not stimulate phospholipase C but engages an unidentified signaling mechanism that promotes transient oscillations in [Ca(2+)](i). We show here that the [Ca(2+)](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, [Ca(2+)](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 [Ca(2+)](i) oscillations can be prevented by protein kinase C (PKC) inhibitors or siRNA-mediated PKCalpha 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 [Ca(2+)](i) oscillations that integrates its stimulation by aromatic amino acids with TRPC1 regulation by PKC and calmodulin.