RasGRP1 Is Essential for Ras Activation by the Tumor Promoter 12-O-Tetradecanoylphorbol-13-acetate in Epidermal Keratinocytes

Amrish Sharma,Courtney T Luke,Nancy A Dower,James C Stone,Patricia S Lorenzo

doi:10.1074/jbc.m109.100016

Amrish Sharma, Courtney T Luke + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m109.100016

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Abstract

RasGRP1 is a guanine nucleotide exchange factor for Ras that binds with high affinity to diacylglycerol analogs like the phorbol esters. Recently, we demonstrated a role for RasGRP1 in skin carcinogenesis and suggested its participation in the action of tumor-promoting phorbol esters like 12-O-tetradecanoylphorbol-13-acetate (TPA) on Ras pathways in epidermal cells. Given the importance of Ras in carcinogenesis, we sought to discern whether RasGRP1 was a critical pathway in Ras activation, using a RasGRP1 knockout (KO) mouse model to examine the response of keratinocytes to TPA. In contrast to the effect seen in wild type keratinocytes, Ras(GTP) levels were barely detected in RasGRP1 KO cells even after 60 min of exposure to phorbol esters. The lack of response was rescued by enforced expression of RasGRP1. Furthermore, small hairpin RNA-induced silencing of RasGRP1 abrogated the effect of TPA on Ras. Analysis of Ras isoforms showed that both H-Ras and N-Ras depended on RasGRP1 for activation by TPA, whereas activation of K-Ras could not be detected. Although RasGRP1 was dispensable for ERK activation in response to TPA, JNK activation was reduced in the KO keratinocytes. Notably, TPA-induced phosphorylation of JNK2, but not JNK1, was reduced by RasGRP1 depletion. These data identify RasGRP1 as a critical molecule in the activation of Ras by TPA in primary mouse keratinocytes and suggest JNK2 as one of the relevant downstream targets. Given the role of TPA as a skin tumor promoter, our findings provide additional support for a role for RasGRP1 in skin carcinogenesis.

Highlights

RasGRP1 is one of the members of the RasGRP family, composed of a total of four isofomors (RasGRP1 to 4) that have different tissue distribution and specificity for Ras-like members (10 –14)
We utilized primary mouse keratinocytes derived from a RasGRP1 knockout (RasGRP1 KO) mouse model as well as an shRNA approach to demonstrate that RasGRP1 is essential for Ras activation in response to tumor-promoting phorbol esters like 12-Otetradecanoylphorbol-13-acetate (TPA)
RasGRP1 Knockout Cells Show Impaired Ras Activation in Response to TPA—To investigate the extent of RasGRP1 contribution to Ras activation by TPA in keratinocytes, we analyzed the response of keratinocytes derived from RasGRP1 KO mouse skin

Summary

To whom correspondence should be addressed

RasGRP1 functions as a guanine nucleotide exchange factor for Ras small GTPases, promoting Ras binding to GTP and Ras activation [15]. It is abundantly expressed in T-cells but is present in other hematopoietic cells and in the brain [14]. We found that keratinocytes derived from the skin express RasGRP1 [9], where it can mediate Ras activation in response to diacylglycerol analogs caused by the presence of a C1-binding domain similar to that of the classic and novel PKC isoforms [9, 16, 17]. Considering that TPA is a potent skin tumor promoter and that JNK pathways are involved in tumorigenic responses in skin [18, 19], our findings may provide insights into the mechanisms of action of tumor promotion and keratinocyte transformation in response to phorbol esters

EXPERIMENTAL PROCEDURES

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DISCUSSION