Ze'ev Ronai, Ph.D.

E3 Ligase Siah and the Hypoxia Response

Our work on the E3 protein ligase Siah led to the identification of a novel layer in regulation of the cellular hypoxia response. We identified prolyl hydroxylases 1 and 3 (PHD1/3) as Siah substrates, in particular, in hypoxia (Nakayama et al., Cell., 2004). These findings establish the mechanism underlying stabilization of HIF1 hypoxia (2-7% oxygen), a level that allows retention of sufficient oxygen for activity of PHDs. These findings also point to the existence of other regulatory components in mild hypoxia, a physiologic state whose role is central in development, differentiation and organ maintenance. More recent studies have pointed out to the role of p38 kinase in the phosphorylation and subcellular localization of Siah2 under hypoxia conditions (Khurana et al., JBC, 2006), and for the assembly of PHD complexes under hypoxia, as a mechanism that affect their activity and susceptibility to Siah2-mediated degradation (Nakayama et al., Biochem. J. 2007). Ongoing studies using Siah2 KO mice points to the importance of Siah2 in tumor development as well as in its metastatic capacity. These findings identify that in melanoma model Siah2 effect on PHD and consequently HIF1a affects the ability of the tumor to metastasize, without affecting its tumorigenic capacity. We have identified that Siah2 effect on Sprouty 2, a negative regulator of Ras signaling pathway, is responsible for tumorigenicity. Thus, Siah2 affect tumor and metastasis in melanoma model via regulation of distinct substrates and regulatory pathways – the Ras signaling for tumor formation (via Sprouty 2) and the HIF signaling (via PHD3) for metastatic capacity. Ongoing studies elucidate the role of Siah2 in prostate tumor model, highlighting novel mechanistic insights into Siah2 regulation and importance in development and progression of different tumor types. The notion that Siah2 is playing such important role in tumor development and progression also prompted screen for inhibitors that would specifically affect this ubiquitin ligase.

E3 Ligase RNF5 and its Associated Protein JAMP – in ER-stress Response

RNF5 is a RING finger E3 ligase which was shown in previous studies from our lab to be involved in regulation of cytoskeletal protein stability as well as subcellular localization. RNF5’s effect on key cytoskeletal proteins affects cell adhesion and motility and is expected to affect tumorigenicity and metastasis capacity, especially in tumors in which it is deregulated. Initial studies revealed role of RNF5 in breast cancer cells organization and proliferation (Broomberg et al., Cancer Res. 2007). Our studies on RNF5 include work in C. elegans, KO mice as well as transgenic mice, which provide important systems that complement our cell biology and biochemical studies. Using transgenic mouse model we discovered that overexpression of RNF5 results in muscular disorder that resembles inclusion Body Myocytis (IBM) – a prevalent muscle disorder in older people which has been associated with extensive ER stress. Our mouse model (rtTA-MCK-RNF5) is the first to allow studying this muscular disorder in mice (Delaunay et al., Plos One, 2008). Ongoing studies point to the role of RNF5 in protein trafficking and degradation, primarily those localized within the ER compartment.

Further, among RNF5-associated proteins is JAMP, a JNK-associated transmembrane protein that affects JNK signaling, which is localized in the ER membrane and emerges as a novel receptor for proteasomes. JAMP recruits proteasomes following ER stress and facilitates the degradation of malfolded proteins (Tcherpakov et al., Mol Biol Cell. 2008). The implications of RNF5 and JAMP activities for ER-stress as well as pathological conditions are currently under investigation using corresponding KO and Tg mouse models.

JNK – Novel Insights into Regulation and Function

Recent studies from our lab identified that the level of JNK activity is regulated by PKC, via the adaptor protein RACK1 (Bergami-Lopez, Mol Cell., 2005). The importance of PKC to JNK signaling is expected to also impact cytoskeletal organization and RNA translation, given the involvement of RACK1 in these processes, aspects that are currently under investigation in our lab. Importantly, JNK activation is subject to changes in pathological cases as in human tumors. Our recent studies in melanoma revealed the mechanism for re-wiring of signal transduction pathways in this tumor type where ERK feeds onto the activation of JNK through upregulation of c-Jun, with concomitant activation of RACK1 – to feed forward PKC-JNK signaling (Bergami Lopez, Cancer Cell, 2007). The implications of such re-wiring are further investigated.

In parallel studies we have identified undisclosed link between JNK and cell cycle control, an aspect that is currently studied, and is expected to shed important new light on the regulation and function of this important protein kinase.

ATF2 - Transcription Factor and DNA Damage Response Protein

Among JNK substrates is ATF2. Our studies have demonstrated the role of the transcription factor ATF2 in the development and notorious resistance of melanomas to treatment. Ongoing studies evaluate chemical compounds for their ability to inhibit melanoma growth and metastatic potential, based on former studies with a 10 or 50aa peptide driven from this transcription factor. Preclinical testing are ongoing with a subset of derivatives which exhibit promising results (Abbas et al., Clin Cancer Res. 2007). Using a mouse model for melanoma we now explore the role of ATF2 in melanoma development using a genetic model. Initial results reveal that lack of ATF2 cause marked inhibition in melanoma development. Mechanisms underlying ATF2 role in melanoma development are currently investigated.

Studies from our lab have also shown that ATF2 is an ATM substrate and that ATF2 functions in the DNA damage response by affecting DSB foci formation and cell cycle checkpoint control. The mechanisms underlying ATF2's contribution to the DNA damage response appear to involve components important for chromatin organization. The implications for ATF2 in tumor development and DNA damage response are currently under investigation using KO and KI mouse models. Using KI ATF2 mouse (where ATF2 phosphorylation sites by ATM were mutated, we identify high sensitivity to IR, resembling ATM mice. Mechanisms underlying ATF2 role in radiation resistance and cell cycle control are currently studied.