Signaling via estrogen receptor (ER) occurs by interacting with many proteins. Nuclear interactome analysis of ERalpha in an embryo implantation model revealed the association of chicken tumor virus no. 10 regulator of kinase like (CrkL) with ERalpha, which was further validated by mammalian two-hybrid assay as well as coimmunoprecipitation and colocalization. Mutation in LPALL motif of CrkL disrupts the ERalpha-CrkL interaction and its transactivation potential, thereby suggesting that the interaction is mediated via its single ER binding motif, Leu-Pro-Ala-Leu-Leu (LXXLL) motif in the sarcoma homology (SH)2 domain. CrkL deletion constructs of SH2 domain target to the nucleus due to presence of nuclear localization signal. Interestingly, the SH2-SH3 (N terminal) construct shows an increased transactivation potential like CrkI. Weak interaction capability of mutated ERalpha-Y538F with CrkL validates that CrkL interacts with ERalpha via its YDLL motif at Tyr 541. In an attempt to understand the physiological relevance of this association, we investigated the impact on cell proliferation using a cancer model, because events associated in the process of pregnancy and cancer are analogous. Also, overexpression of CrkL is frequently associated with tumorigenesis. However, its significance in hormone-regulated cancers still remains obscure. Here, we demonstrate that association of ERalpha and CrkL directly enhances the tumorigenic potential of CrkL, thus pointing to its role in cell proliferation. In human endometrial cancers, we observed a strong association between ERalpha and CrkL levels. Thus, the molecular signaling set off by ERalpha and CrkL association may have a central role in pregnancy and cancer, two events which share parallels in growth, invasion, and immune tolerance.
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