RESUMEN
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.
Asunto(s)
Membrana Celular/enzimología , Núcleo Celular/enzimología , Proteína Quinasa C/metabolismo , Quinasas p21 Activadas/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Transporte Activo de Núcleo Celular/genética , Animales , Línea Celular , Membrana Celular/genética , Núcleo Celular/genética , Ratones , Fosforilación/efectos de los fármacos , Fosforilación/genética , Proteína Quinasa C/genética , Inhibidores de Proteínas Quinasas/farmacología , Ratas , Quinasas p21 Activadas/antagonistas & inhibidores , Quinasas p21 Activadas/genéticaRESUMEN
We examined the regulation of Yes-associated protein (YAP) localization, phosphorylation, and transcriptional activity in intestinal epithelial cells. Our results show that stimulation of intestinal epithelial IEC-18 cells with the G protein-coupled receptor (GPCR) agonist angiotensin II, a potent mitogen for these cells, induced rapid translocation of YAP from the nucleus to the cytoplasm (within 15 min) and a concomitant increase in YAP phosphorylation at Ser(127) and Ser(397) Angiotensin II elicited YAP phosphorylation and cytoplasmic accumulation in a dose-dependent manner (ED50 = 0.3 nm). Similar YAP responses were provoked by stimulation with vasopressin or serum. Treatment of the cells with the protein kinase D (PKD) family inhibitors CRT0066101 and kb NB 142-70 prevented the increase in YAP phosphorylation on Ser(127) and Ser(397) via Lats2, YAP cytoplasmic accumulation, and increase in the mRNA levels of YAP/TEAD-regulated genes (Ctgf and Areg). Furthermore, siRNA-mediated knockdown of PKD1, PKD2, and PKD3 markedly attenuated YAP nuclear-cytoplasmic shuttling, phosphorylation at Ser(127), and induction of Ctgf and Areg expression in response to GPCR activation. These results identify a novel role for the PKD family in the control of biphasic localization, phosphorylation, and transcriptional activity of YAP in intestinal epithelial cells. In turn, YAP and TAZ are necessary for the stimulation of the proliferative response of intestinal epithelial cells to GPCR agonists that act via PKD. The discovery of interaction between YAP and PKD pathways identifies a novel cross-talk in signal transduction and demonstrates, for the first time, that the PKDs feed into the YAP pathway.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Enterocitos/metabolismo , Proteína Quinasa C/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Transducción de Señal , Aciltransferasas , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/genética , Línea Celular , Núcleo Celular/genética , Citoplasma/genética , Compuestos Heterocíclicos con 3 Anillos/farmacología , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fosforilación/efectos de los fármacos , Fosforilación/genética , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/genética , Pirimidinas/farmacología , Ratas , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Tiazepinas/farmacología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Señalizadoras YAPRESUMEN
Given the fundamental role of ß-catenin signaling in intestinal epithelial cell proliferation and the growth-promoting function of protein kinase D1 (PKD1) in these cells, we hypothesized that PKDs mediate cross talk with ß-catenin signaling. The results presented here provide several lines of evidence supporting this hypothesis. We found that stimulation of intestinal epithelial IEC-18 cells with the G protein-coupled receptor (GPCR) agonist angiotensin II (ANG II), a potent inducer of PKD activation, promoted endogenous ß-catenin nuclear localization in a time-dependent manner. A significant increase was evident within 1 h of ANG II stimulation (P< 0.01), peaked at 4 h (P< 0.001), and declined afterwards. GPCR stimulation also induced a marked increase in ß-catenin-regulated genes and phosphorylation at Ser(552) in intestinal epithelial cells. Exposure to preferential inhibitors of the PKD family (CRT006610 or kb NB 142-70) or knockdown of the isoforms of the PKD family prevented the increase in ß-catenin nuclear localization and phosphorylation at Ser(552) in response to ANG II. GPCR stimulation also induced the formation of a complex between PKD1 and ß-catenin, as shown by coimmunoprecipitation that depended on PKD1 catalytic activation, as it was abrogated by cell treatment with PKD family inhibitors. Using transgenic mice that express elevated PKD1 protein in the intestinal epithelium, we detected a marked increase in the localization of ß-catenin in the nucleus of crypt epithelial cells in the ileum of PKD1 transgenic mice, compared with nontransgenic littermates. Collectively, our results identify a novel cross talk between PKD and ß-catenin in intestinal epithelial cells, both in vitro and in vivo.
Asunto(s)
Núcleo Celular/metabolismo , Mucosa Intestinal/metabolismo , Proteína Quinasa C/metabolismo , Receptor Cross-Talk/fisiología , beta Catenina/metabolismo , Transporte Activo de Núcleo Celular , Animales , Línea Celular , Técnica del Anticuerpo Fluorescente , Técnicas de Silenciamiento del Gen , Humanos , Procesamiento de Imagen Asistido por Computador , Immunoblotting , Inmunoprecipitación , Ratones , Ratones Transgénicos , Fosforilación , ARN Interferente Pequeño , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Serina/metabolismo , TransfecciónRESUMEN
Cushing disease (CD) is a life-threatening disorder attributed to excess pituitary tumor-derived adrenocorticotrophic hormone (ACTH) and adrenal steroid secretion caused by pituitary tumors. Whereas CD was first described in 1932, the underlying genetic basis driving tumor growth and ACTH secretion remains unsolved. Here, we show that testicular orphan nuclear receptor 4 (TR4, nuclear receptor subfamily 2, group C, member 2) is overexpressed in human corticotroph tumors as well as in human and mouse corticotroph tumor cell lines. Forced overexpression of TR4 in both human and murine tumor cells increased proopiomelanocortin transcription, ACTH secretion, cellular proliferation, and tumor invasion rates in vitro. Conversely, knockdown of TR4 expression reversed all phenotypes. Mechanistically, we show that TR4 transcriptionally activates proopiomelanocortin through binding of a direct repeat 1 response element in the promoter, and that this is enhanced by MAPK-mediated TR4 phosphorylation. In vivo, TR4 overexpression promotes murine corticotroph tumor growth as well as enhances ACTH and corticosterone production, whereas TR4 knockdown decreases circulating ACTH and corticosterone levels in mice harboring ACTH-secreting tumors. Our findings directly link TR4 to the etiology of corticotroph tumors, hormone secretion, and cell growth as well as identify it as a potential target in the treatment of CD.
Asunto(s)
Adenoma Hipofisario Secretor de ACTH/metabolismo , Hormona Adrenocorticotrópica/metabolismo , Proteínas de Neoplasias/metabolismo , Hipersecreción de la Hormona Adrenocorticotrópica Pituitaria (HACT)/metabolismo , Receptores de Esteroides/metabolismo , Receptores de Hormona Tiroidea/metabolismo , Adenoma Hipofisario Secretor de ACTH/genética , Adenoma Hipofisario Secretor de ACTH/patología , Adenoma Hipofisario Secretor de ACTH/terapia , Hormona Adrenocorticotrópica/genética , Animales , Línea Celular Tumoral , Proliferación Celular , Corticosterona/genética , Corticosterona/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Ratones , Proteínas de Neoplasias/genética , Hipersecreción de la Hormona Adrenocorticotrópica Pituitaria (HACT)/genética , Hipersecreción de la Hormona Adrenocorticotrópica Pituitaria (HACT)/patología , Hipersecreción de la Hormona Adrenocorticotrópica Pituitaria (HACT)/terapia , Proopiomelanocortina/biosíntesis , Proopiomelanocortina/genética , Receptores de Esteroides/genética , Receptores de Hormona Tiroidea/genética , Elementos de Respuesta/genética , Activación Transcripcional/genéticaRESUMEN
Obesity, a known risk factor for pancreatic cancer, is associated with inflammation and insulin resistance. Proinflammatory prostaglandin E2 (PGE2) and elevated insulin-like growth factor type 1 (IGF-1), related to insulin resistance, are shown to play critical roles in pancreatic cancer progression. We aimed to explore a potential cross talk between PGE2 signaling and the IGF-1/Akt/mammalian target of rapamycin complex 1 (mTORC1) pathway in pancreatic cancer, which may be a key to unraveling the obesity-cancer link. In PANC-1 human pancreatic cancer cells, we showed that PGE2 stimulated mTORC1 activity independently of Akt, as evaluated by downstream signaling events. Subsequently, using pharmacological and genetic approaches, we demonstrated that PGE2-induced mTORC1 activation is mediated by the EP4/cAMP/PKA pathway, as well as an EP1/Ca(2+)-dependent pathway. The cooperative roles of the two pathways were supported by the maximal inhibition achieved with the combined pharmacological blockade, and the coexistence of highly expressed EP1 (mediating the Ca(2+) response) and EP2 or EP4 (mediating the cAMP/PKA pathway) in PANC-1 cells and in the prostate cancer line PC-3, which also robustly exhibited PGE2-induced mTORC1 activation, as identified from a screen in various cancer cell lines. Importantly, we showed a reinforcing interaction between PGE2 and IGF-1 on mTORC1 signaling, with an increase in IL-23 production as a cellular outcome. Our data reveal a previously unrecognized mechanism of PGE2-stimulated mTORC1 activation mediated by EP4/cAMP/PKA and EP1/Ca(2+) signaling, which may be of great importance in elucidating the promoting effects of obesity in pancreatic cancer. Ultimately, a precise understanding of these molecular links may provide novel targets for efficacious interventions devoid of adverse effects.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Dinoprostona/farmacología , Complejos Multiproteicos/metabolismo , Subtipo EP1 de Receptores de Prostaglandina E/metabolismo , Subtipo EP4 de Receptores de Prostaglandina E/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Calcio/metabolismo , Línea Celular Tumoral , AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Dinoprostona/metabolismo , Regulación de la Expresión Génica/fisiología , Humanos , Subunidad p19 de la Interleucina-23/genética , Subunidad p19 de la Interleucina-23/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina , Complejos Multiproteicos/genética , Subtipo EP1 de Receptores de Prostaglandina E/genética , Subtipo EP2 de Receptores de Prostaglandina E/genética , Subtipo EP2 de Receptores de Prostaglandina E/metabolismo , Subtipo EP4 de Receptores de Prostaglandina E/genética , Serina-Treonina Quinasas TOR/genéticaRESUMEN
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.
Asunto(s)
Señalización del Calcio , Receptores Sensibles al Calcio/genética , Receptores Sensibles al Calcio/metabolismo , Sustitución de Aminoácidos , Sitios de Unión/genética , Calcio/metabolismo , Calcio/farmacología , Señalización del Calcio/efectos de los fármacos , Células HEK293 , Humanos , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fenilalanina/farmacología , Estructura Terciaria de Proteína , Receptores Sensibles al Calcio/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
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).
Asunto(s)
Señalización del Calcio , Calcio/metabolismo , Proteína Quinasa C-alfa/metabolismo , Receptores Sensibles al Calcio/metabolismo , Compuestos de Anilina/farmacología , Calcio/agonistas , Línea Celular , Inhibidores Enzimáticos/farmacología , Células HEK293 , Humanos , Indoles/farmacología , Transporte Iónico , Maleimidas/farmacología , Fenetilaminas , Fosforilación , Propilaminas , Proteína Quinasa C-alfa/antagonistas & inhibidores , Proteína Quinasa C-alfa/genética , Inhibidores de Proteínas Quinasas/farmacología , Interferencia de ARN , ARN Interferente PequeñoRESUMEN
We examined whether class IIa histone deacetylases (HDACs) play a role in mitogenic signaling mediated by protein kinase D1 (PKD1) in IEC-18 intestinal epithelial cells. Our results show that class IIa HDAC4, HDAC5, and HDAC7 are prominently expressed in these cells. Stimulation with ANG II, a potent mitogen for IEC-18 cells, induced a striking increase in phosphorylation of HDAC4 at Ser(246) and Ser(632), HDAC5 at Ser(259) and Ser(498), and HDAC7 at Ser(155). Treatment with the PKD family inhibitors kb NB 142-70 and CRT0066101 or small interfering RNA-mediated knockdown of PKD1 prevented ANG II-induced phosphorylation of HDAC4, HDAC5, and HDAC7. A variety of PKD1 activators in IEC-18 cells, including vasopressin, lysophosphatidic acid, and phorbol esters, also induced HDAC4, HDAC5, and HDAC7 phosphorylation. Using endogenously and ectopically expressed HDAC5, we show that PKD1-mediated phosphorylation of HDAC5 induces its nuclear extrusion into the cytoplasm. In contrast, HDAC5 with Ser(259) and Ser(498) mutated to Ala was localized to the nucleus in unstimulated and stimulated cells. Treatment of IEC-18 cells with specific inhibitors of class IIa HDACs, including MC1568 and TMP269, prevented cell cycle progression, DNA synthesis, and proliferation induced in response to G protein-coupled receptor/PKD1 activation. The PKD1-class IIa HDAC axis also functions in intestinal epithelial cells in vivo, since an increase in phosphorylation of HDAC4/5 and HDAC7 was demonstrated in lysates of crypt cells from PKD1 transgenic mice compared with matched nontransgenic littermates. Collectively, our results reveal a PKD1-class IIa HDAC axis in intestinal epithelial cells leading to mitogenic signaling.
Asunto(s)
Células Epiteliales/enzimología , Histona Desacetilasas/metabolismo , Mucosa Intestinal/enzimología , Proteína Quinasa C/metabolismo , Transducción de Señal/genética , Angiotensina II/farmacología , Animales , Línea Celular , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Regulación de la Expresión Génica , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/genética , Mucosa Intestinal/citología , Mucosa Intestinal/efectos de los fármacos , Isoenzimas/genética , Isoenzimas/metabolismo , Lisofosfolípidos/farmacología , Ratones , Ratones Transgénicos , Ésteres del Forbol/farmacología , Fosforilación , Proteína Quinasa C/genética , Transporte de Proteínas , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ratas , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Vasopresinas/farmacologíaRESUMEN
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.
Asunto(s)
Núcleo Celular/metabolismo , Colon/metabolismo , Mucosa Intestinal/metabolismo , Receptores Sensibles al Calcio/metabolismo , Transcripción Genética/fisiología , beta Catenina/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Transporte Activo de Núcleo Celular/fisiología , Compuestos de Anilina/farmacología , Animales , Calcio/metabolismo , Calcio/farmacología , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Línea Celular , Núcleo Celular/genética , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Humanos , Ratones , Ratones Noqueados , Fenetilaminas , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Propilaminas , Receptores Sensibles al Calcio/genética , Transcripción Genética/efectos de los fármacos , beta Catenina/genéticaRESUMEN
Following transplantation, HLA class I antibodies targeting donor endothelium stimulate cell proliferation and migration, which contribute to the development of transplant vasculopathy and chronic allograft rejection. Dynamic remodeling of the actin cytoskeleton regulates cell proliferation and migration in endothelial cells (ECs), but the mechanism(s) involved remain incompletely understood. We explored anti-HLA class I antibody-mediated alterations of the cytoskeleton in human aortic ECs (HAECs) and contrasted these findings to thrombin-induced cytoskeleton remodeling. Our results identify two different signaling pathways leading to myosin light chain (MLC) phosphorylation in HAECs. Stimulation of HAECs with thrombin at 1 U/ml induced a robust elevation of intracellular Ca(2+) concentration, increased MLC phosphorylation, and promoted stress fiber formation via MLC kinase (MLCK) and Rho kinase (ROK) in an ERK-independent manner. In contrast, HAECs stimulated with HLA class I antibodies did not promote any detectable change in intracellular Ca(2+) concentration but instead induced MLC phosphorylation and stress fiber assembly via MLCK and ROK in an ERK1/2-dependent manner. Stimulation of HAECs with low-dose thrombin (1 mU/ml) induced signaling cascades that were similar to stimulation with HLA class I antibodies. HLA class I antibodies also stimulated the translocation of mammalian target of rapamycin complex 2 (mTORC2) and ERK1/2 from the cytoplasm to the plasma membrane independently of stress fiber assembly. These findings identify novel roles for HLA class I signaling in ECs and provide new insights into the role of ERK1/2 and mTORC2 in cytoskeleton regulation, which may be important in promoting transplant vasculopathy, tumor angiogenesis, and atherosclerosis.
Asunto(s)
Aorta/citología , Aorta/metabolismo , Calcio/metabolismo , Células Endoteliales/enzimología , Antígenos de Histocompatibilidad Clase I/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fibras de Estrés/metabolismo , Línea Celular , Células Endoteliales/citología , Trasplante de Corazón , Humanos , Hibridomas , Sistema de Señalización de MAP Quinasas/fisiología , Diana Mecanicista del Complejo 2 de la Rapamicina , Complejos Multiproteicos/metabolismo , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
We have examined the role of protein kinase D1 (PKD1) signaling in intestinal epithelial cell migration. Wounding monolayer cultures of intestinal epithelial cell line IEC-18 or IEC-6 induced rapid PKD1 activation in the cells immediately adjacent to the wound edge, as judged by immunofluorescence microscopy with an antibody that detects the phosphorylated state of PKD1 at Ser(916), an autophosphorylation site. An increase in PKD1 phosphorylation at Ser(916) was evident as early as 45 s after wounding, reached a maximum after 3 min, and persisted for ≥15 min. PKD1 autophosphorylation at Ser(916) was prevented by the PKD family inhibitors kb NB 142-70 and CRT0066101. A kb NB 142-70-sensitive increase in PKD autophosphorylation was also elicited by wounding IEC-6 cells. Using in vitro kinase assays after PKD1 immunoprecipitation, we corroborated that wounding IEC-18 cells induced rapid PKD1 catalytic activation. Further results indicate that PKD1 signaling is required to promote migration of intestinal epithelial cells into the denuded area of the wound. Specifically, treatment with kb NB 142-70 or small interfering RNAs targeting PKD1 markedly reduced wound-induced migration in IEC-18 cells. To test whether PKD1 promotes migration of intestinal epithelial cells in vivo, we used transgenic mice that express elevated PKD1 protein in the small intestinal epithelium. Enterocyte migration was markedly increased in the PKD1 transgenic mice. These results demonstrate that PKD1 activation is one of the early events initiated by wounding a monolayer of intestinal epithelial cells and indicate that PKD1 signaling promotes the migration of these cells in vitro and in vivo.
Asunto(s)
Movimiento Celular/fisiología , Células Epiteliales/fisiología , Proteína Quinasa C/metabolismo , Cicatrización de Heridas/fisiología , Animales , Línea Celular , Células Cultivadas , Activación Enzimática , Ratones , Ratones Transgénicos , Fosforilación , Proteína Quinasa C/antagonistas & inhibidores , Pirimidinas/farmacología , Transducción de SeñalRESUMEN
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.
Asunto(s)
Sistema de Transporte de Aminoácidos A/metabolismo , Aminoácidos/metabolismo , Señalización del Calcio , Células Enteroendocrinas/metabolismo , Sistema de Transporte de Aminoácidos A/genética , Animales , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/efectos de los fármacos , Canales de Calcio Tipo L/metabolismo , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/genética , Línea Celular Tumoral , Células Enteroendocrinas/efectos de los fármacos , Humanos , Activación del Canal Iónico , Potenciales de la Membrana , Ratones , Nifedipino/farmacología , Nitrendipino/farmacología , Interferencia de ARN , Receptores Sensibles al Calcio/genética , Receptores Sensibles al Calcio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sodio/metabolismo , Factores de Tiempo , TransfecciónRESUMEN
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.
Asunto(s)
Señalización del Calcio/fisiología , Calcio/metabolismo , Proliferación Celular , Colon/citología , Células Epiteliales/fisiología , Receptores Sensibles al Calcio/metabolismo , Actinas/metabolismo , Línea Celular , Proteínas Contráctiles/metabolismo , Citoesqueleto/metabolismo , Células Epiteliales/citología , Filaminas , Humanos , Proteínas de Microfilamentos/metabolismo , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Sensibles al Calcio/genética , Canales Catiónicos TRPC/metabolismo , Proteínas de Unión al GTP rho/metabolismoRESUMEN
We examined crosstalk between the insulin receptor and G protein-coupled receptor (GPCR) signaling pathways in individual human pancreatic cancer PANC-1 cells. Treatment of cells with insulin (10 ng/ml) for 5 min markedly enhanced the proportion of cells that display an increase in intracellular [Ca²+] induced by picomolar concentrations of the GPCR agonist neurotensin. Interestingly, insulin increased the proportion of a subpopulation of cells that exhibit intracellular [Ca²+] oscillations in response to neurotensin at concentrations as low as 50-200 pM. Insulin enhanced GPCR-induced Ca²+ signaling in a time- and dose-dependent manner; a marked potentiation was obtained after an exposure to a concentration of 10 ng/ml for 5 min. Treatment with the mTORC1 inhibitor rapamycin abrogated the increase in GPCR-induced [Ca²+](i) oscillations produced by insulin. Our results identify a novel aspect in the crosstalk between insulin receptor and GPCR signaling systems in pancreatic cancer cells, namely that insulin increases the number of [Ca²+](i) oscillating cells induced by physiological concentrations of GPCR agonists through an mTORC1-dependent pathway.
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Señalización del Calcio , Calcio/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Neoplasias Pancreáticas/metabolismo , Receptor de Insulina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Línea Celular Tumoral , Humanos , Insulina/metabolismo , Insulina/farmacología , Diana Mecanicista del Complejo 1 de la Rapamicina , Complejos Multiproteicos , Neurotensina/metabolismo , Neurotensina/farmacología , Proteínas , Serina-Treonina Quinasas TOR , Factores de Transcripción/metabolismoRESUMEN
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.
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Insulina/farmacología , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteínas Quinasas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Angiotensina II/farmacología , Western Blotting , Bombesina/farmacología , Bradiquinina/farmacología , Señalización del Calcio/efectos de los fármacos , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Humanos , Hidrólisis/efectos de los fármacos , Inositol 1,4,5-Trifosfato/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Microscopía Fluorescente , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada , Neurotensina/farmacología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Fosforilación/efectos de los fármacos , Receptores Acoplados a Proteínas G/agonistas , Sirolimus/farmacología , Serina-Treonina Quinasas TOR , Vasopresinas/farmacologíaRESUMEN
We examined the impact of crosstalk between the insulin receptor and G protein-coupled receptor (GPCR) signaling pathways on the regulation of Yes-associated protein (YAP) localization, phosphorylation, and transcriptional activity in the context of human pancreatic ductal adenocarcinoma (PDAC). Stimulation of PANC-1 or MiaPaCa-2 cells with insulin and neurotensin, a potent mitogenic combination of agonists for these cells, promoted striking YAP nuclear localization and decreased YAP phosphorylation at Ser127 and Ser397 Challenging PDAC cells with either insulin or neurotensin alone modestly induced the expression of YAP/TEAD-regulated genes, including connective tissue growth factor (CTGF), cysteine-rich angiogenic inducer 61 (CYR61), and CXCL5, whereas the combination of neurotensin and insulin induced a marked increase in the level of expression of these genes. In addition, siRNA-mediated knockdown of YAP/TAZ prevented the increase in the expression of these genes. A small-molecule inhibitor (A66), selective for the p110α subunit of PI3K, abrogated the increase in phosphatidylinositol 3,4,5-trisphosphate production and the expression of CTGF, CYR61, and CXCL5 induced by neurotensin and insulin. Furthermore, treatment of PDAC cells with protein kinase D (PKD) family inhibitors (CRT0066101 or kb NB 142-70) or with siRNAs targeting the PKD family prevented the increase of CTGF, CYR61, and CXCL5 mRNA levels in response to insulin and neurotensin stimulation. Thus, PI3K and PKD mediate YAP activation in response to insulin and neurotensin in pancreatic cancer cells.Implications: Inhibitors of PI3K or PKD disrupt crosstalk between insulin receptor and GPCR signaling systems by blocking YAP/TEAD-regulated gene expression in pancreatic cancer cells. Mol Cancer Res; 15(7); 929-41. ©2017 AACR.
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Proteínas Adaptadoras Transductoras de Señales/genética , Carcinoma Ductal Pancreático/genética , Fosfatidilinositol 3-Quinasa Clase I/genética , Neoplasias Pancreáticas/genética , Fosfoproteínas/genética , Proteína Quinasa C/genética , Antígenos CD/genética , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Quimiocina CXCL5/genética , Factor de Crecimiento del Tejido Conjuntivo/genética , Proteína 61 Rica en Cisteína/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Insulina/administración & dosificación , Insulina/metabolismo , Neurotensina/administración & dosificación , Neurotensina/metabolismo , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/patología , Fosforilación/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Pirimidinas/administración & dosificación , Receptor de Insulina/genética , Receptores Acoplados a Proteínas G/genética , Factores de Transcripción , Proteínas Señalizadoras YAPRESUMEN
The PI3K/AKT/mTOR pathway, which is aberrantly stimulated in many cancer cells, has emerged as a target for therapy. However, mTORC1/S6K also mediates negative feedback loops that attenuate upstream signaling. Suppression of these feedback loops opposes the growth-suppressive effects of mTOR inhibitors and leads to drug resistance. Here, we demonstrate that treatment of PANC-1 or MiaPaCa-2 pancreatic ductal adenocarcinoma (PDAC) cells with the dual PI3K/mTOR kinase inhibitor (PI3K/TOR-KI) BEZ235 blocked mTORC1/S6K activation (scored by S6 phosphorylation at Ser(240/244)), mTORC1/4E-BP1 (assayed by 4E-BP1 phosphorylation at Thr(37/46)), and mTORC2-mediated AKT phosphorylation at Ser(473), in a concentration-dependent manner. Strikingly, BEZ235 markedly enhanced the MEK/ERK pathway in a dose-dependent manner. Maximal ERK overactivation coincided with complete inhibition of phosphorylation of AKT and 4E-BP1. ERK overactivation was induced by other PI3K/TOR-KIs, including PKI-587 and GDC-0980. The MEK inhibitors U126 or PD0325901 prevented ERK overactivation induced by PI3K/TOR-KIs. The combination of BEZ235 and PD0325901 caused a more pronounced inhibition of cell growth than that produced by each inhibitor individually. Mechanistic studies assessing PI3K activity in single PDAC cells indicate that PI3K/TOR-KIs act through a PI3K-independent pathway. Doses of PI3K/TOR-KIs that enhanced MEK/ERK activation coincided with those that inhibited mTORC2-mediated AKT phosphorylation on Ser(473), suggesting a role of mTORC2. Knockdown of RICTOR via transfection of siRNA markedly attenuated the enhancing effect of BEZ235 on ERK phosphorylation. We propose that dual PI3K/mTOR inhibitors suppress a novel negative feedback loop mediated by mTORC2, thereby leading to enhanced MEK/ERK pathway activity in pancreatic cancer cells.
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Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Complejos Multiproteicos/antagonistas & inhibidores , Neoplasias Pancreáticas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Proteínas Portadoras/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Sinergismo Farmacológico , Receptores ErbB , Técnicas de Silenciamiento del Gen , Humanos , Imidazoles/farmacología , Diana Mecanicista del Complejo 2 de la Rapamicina , Morfolinas/farmacología , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Pirimidinas/farmacología , Quinolinas/farmacología , Proteína Asociada al mTOR Insensible a la Rapamicina , Receptor IGF Tipo 1 , Receptor de Insulina/metabolismo , Receptores de Somatomedina/metabolismo , Triazinas/farmacologíaRESUMEN
Natural products represent a rich reservoir of potential small chemical molecules exhibiting anti-proliferative and chemopreventive properties. Here, we show that treatment of pancreatic ductal adenocarcinoma (PDAC) cells (PANC-1, MiaPaCa-2) with the isoquinoline alkaloid berberine (0.3-6 µM) inhibited DNA synthesis and proliferation of these cells and delay the progression of their cell cycle in G1. Berberine treatment also reduced (by 70%) the growth of MiaPaCa-2 cell growth when implanted into the flanks of nu/nu mice. Mechanistic studies revealed that berberine decreased mitochondrial membrane potential and intracellular ATP levels and induced potent AMPK activation, as shown by phosphorylation of AMPK α subunit at Thr-172 and acetyl-CoA carboxylase (ACC) at Ser79. Furthermore, berberine dose-dependently inhibited mTORC1 (phosphorylation of S6K at Thr389 and S6 at Ser240/244) and ERK activation in PDAC cells stimulated by insulin and neurotensin or fetal bovine serum. Knockdown of α1 and α2 catalytic subunit expression of AMPK reversed the inhibitory effect produced by treatment with low concentrations of berberine on mTORC1, ERK and DNA synthesis in PDAC cells. However, at higher concentrations, berberine inhibited mitogenic signaling (mTORC1 and ERK) and DNA synthesis through an AMPK-independent mechanism. Similar results were obtained with metformin used at doses that induced either modest or pronounced reductions in intracellular ATP levels, which were virtually identical to the decreases in ATP levels obtained in response to berberine. We propose that berberine and metformin inhibit mitogenic signaling in PDAC cells through dose-dependent AMPK-dependent and independent pathways.
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Proteínas Quinasas Activadas por AMP/metabolismo , Adenocarcinoma/tratamiento farmacológico , Berberina/farmacología , Carcinoma Ductal Pancreático/tratamiento farmacológico , Proliferación Celular/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Hipoglucemiantes/farmacología , Metformina/farmacología , Complejos Multiproteicos/antagonistas & inhibidores , Neoplasias Pancreáticas/tratamiento farmacológico , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Proteínas Quinasas Activadas por AMP/genética , Acetil-CoA Carboxilasa/metabolismo , Adenocarcinoma/patología , Animales , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Humanos , Insulina/farmacología , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Neurotensina/farmacología , Neoplasias Pancreáticas/patología , Fosforilación/efectos de los fármacos , Interferencia de ARN , ARN Interferente Pequeño/genética , Transducción de Señal/efectos de los fármacos , Trasplante HeterólogoRESUMEN
We examined whether protein kinase D1 (PKD1) mediates negative feeback of PI3K/Akt signaling in intestinal epithelial cells stimulated with G protein-coupled receptor (GPCR) agonists. Exposure of intestinal epithelial IEC-18 cells to increasing concentrations of the PKD family inhibitor kb NB 142-70, at concentrations that inhibited PKD1 activation, strikingly potentiated Akt phosphorylation at Thr(308) and Ser(473) in response to the mitogenic GPCR agonist angiotensin II (ANG II). Enhancement of Akt activation by kb NB 142-70 was also evident in cells with other GPCR agonists, including vasopressin and lysophosphatidic acid. Cell treatment with the structurally unrelated PKD family inhibitor CRT0066101 increased Akt phosphorylation as potently as kb NB 142-70 [corrected]. Knockdown of PKD1 with two different siRNAs strikingly enhanced Akt phosphorylation in response to ANG II stimulation in IEC-18 cells. To determine whether treatment with kb NB 142-70 enhances accumulation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) in the plasma membrane, we monitored the redistribution of Akt-pleckstrin homology domain-green fluorescent protein (Akt-PH-GFP) in single IEC-18 cells. Exposure to kb NB 142-70 strikingly increased membrane accumulation of Akt-PH-GFP in response to ANG II. The translocation of the PIP3 sensor to the plasma membrane and the phosphorylation of Akt was completed prevented by prior exposure to the class I p110α specific inhibitor A66. ANG II markedly increased the phosphorylation of p85α detected by a PKD motif-specific antibody and enhanced the association of p85α with PTEN. Transgenic mice overexpressing PKD1 showed a reduced phosphorylation of Akt at Ser(473) in intestinal epithelial cells compared to wild type littermates. Collectively these results indicate that PKD1 activation mediates feedback inhibition of PI3K/Akt signaling in intestinal epithelial cells in vitro and in vivo.
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Retroalimentación Fisiológica , Fosfatidilinositol 3-Quinasas/metabolismo , Proteína Quinasa C/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Angiotensina II/farmacología , Animales , Línea Celular , Membrana Celular/metabolismo , Fosfatidilinositol 3-Quinasa Clase Ia/metabolismo , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Expresión Génica , Técnicas de Silenciamiento del Gen , Mucosa Intestinal/metabolismo , Ratones , Ratones Transgénicos , Fosfohidrolasa PTEN/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Unión Proteica , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/genética , Inhibidores de Proteínas Quinasas/farmacología , Transporte de Proteínas/efectos de los fármacos , Ratas , Receptores Acoplados a Proteínas G/agonistasRESUMEN
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.