SOS1 is subject to opinions phosphorylation by ERK or ERKRsk at multiple sites, and ERK-regulated phosphorylation negatively modulates SOS1 conversation with Grb2, Shc, and the EGF receptor to dampen growth factor signaling (57). differs from EGF signaling in that it is independent of the Ras guanine nucleotide exchange factors (Ras-GEFs), SOS1/2, and entails prolonged rather than transient ERK activation. PKC forms complexes with A-Raf, B-Raf, and C-Raf that dissociate upon pathway activation, and all three Raf isoforms can mediate PKC-induced antiproliferative effects. At least two PKCCERK pathways that collaborate to promote growth arrest were recognized: one pathway requiring the Ras-GEF, RasGRP3, and H-Ras, prospects to p21Cip1 upregulation, while additional pathway(s) mediate PKC-induced cyclin D1 and Id1 downregulation. PKC also induces ERK-dependent SOS1 phosphorylation, indicating possible unfavorable crosstalk between antiproliferative and growth-promoting ERK signaling. Importantly, the spatiotemporal activation of PKC and ERK in the intestinal epithelium supports the physiological relevance of these pathways and Dasatinib Monohydrate highlights the importance of antiproliferative ERK signaling to tissue homeostasis in the intestine. analysis using nontransformed intestinal epithelial cells confirmed that PKC activation triggers a program of cell cycle withdrawal, including downregulation of pro-proliferative cyclin D1 and inhibitor of DNA-binding 1 (Id1) and upregulation of the cyclin-dependent kinase (CDK) inhibitor p21Cip1 (9, 10, 26, 27, 28). Notably, these Dasatinib Monohydrate effects are dependent on MEK activation (11), indicating that PKC initiates growth inhibitory ERK signaling in intestinal Gadd45a epithelial cells. While it has long Dasatinib Monohydrate been acknowledged that PKC signaling can activate the ERK pathway, there is controversy regarding the mechanism involved. While some studies have indicated that PKC activation of the pathway is usually Ras impartial (29) and can be mediated by direct phosphorylation of C-Raf by PKC (30), other studies indicate that Ras is required (31). Previous studies also support the ability of classical PKCs to activate ERK through a mechanism that does not require Raf Dasatinib Monohydrate phosphorylation (32) and that PKC may directly activate MEK (33). Thus, mechanisms underlying PKC-mediated activation of ERK remain to be elucidated. In the present study, we define crosstalk between PKC and the ERK activation cascade during growth suppressive signaling in intestinal epithelial cells. Our analysis has decided that PKC intersects the canonical ERK activation pathway at the level of Ras and that PKC triggers at least two?antiproliferative ERK signaling pathways. One pathway entails activation of RasGRP3 and H-Ras for ERK-dependent?induction of p21Cip1, while at least one other PKCCRasCERK pathway triggers downregulation of the pro-proliferative proteins, cyclin D1 and Id1. Results Activation of PKC by PMA induces ERK-dependent antiproliferative signaling in intestinal epithelial cells We have previously reported that pharmacological activation of PKC in nontransformed IEC-18 intestinal crypt-like cells induces cell cycle arrest in association with activation of the ERK signaling pathway, as decided using antiphospho-ERK (pERK) immunoblotting (ref. (11) and Fig.?1of the blots shows densitometric analysis of relative levels of pERK normalized to loading control (SD, n?= 5, ? 0.05; ?? 0.01); ii, IEC-18?cells were treated with PMA for 6?h in the absence or presence of 4?M?G?6976 and analyzed for the expression of PKC, , or by Western blotting. are therefore the same in both figures. and and and and and and and are not required for and and of the blots in ( 0.05; ?? 0.01). All data in (show rearrangement of the blot for clarity. signaling may promote ERK-dependent opinions inhibition of While our analysis recognized SOS1/2-impartial growth suppressive PKC-ERK signaling, the data did not exclude the possibility that antiproliferative PKC-ERK signaling may also intersect growth-promoting ERK pathways through inhibition of SOS Dasatinib Monohydrate proteins. ERK-dependent phosphorylation of SOS1 is usually a major mechanism for negative opinions of growth factorCinduced Ras-ERK signaling. ERK phosphorylates SOS1 on Ser1132, Ser1167, Ser1178, and Ser1193 in the C-terminal domain name, which inhibits SOS1 function by reducing its association with Grb2, Shc, and the EGF receptor (51, 52, 53). Unfavorable opinions of Ras activation can also result from ERKRsk-mediated phosphorylation of SOS1 (and ?and99 0.05). iii, as in (ii), except that cells were treated with PMA for 2?h. are included for clarity. indicates rearrangement of the blot for clarity. ii, IEC-18?cells were pretreated with 4?M?G?6976 to inhibit PKC activity, followed by 15?min treatment with 100?nM PMA as indicated. Cells were then lysed and lysates were divided and treated with lambda phosphatase.