Baccarini

RAF1 and MEK1 are moonlighting proteins acting as signaling hubs to coordinate ERK activation with the control of other signaling pathways

BRAF is the ancestral RAF kinase and the main MEK/ERK activator in vivo. Phenotypes connected with its ablation are caused, and can be phenocopied, by reduced ERK activation (2). In contrast, RAF1 and most likely ARAF have acquired additional essential functions (2). RAF1 promotes survival independently of its enzymatic activity by binding to and negatively regulating the pro-apoptotic kinases [MST2 (3,4) and ASK1 (5)] and the RHO-dependent kinase ROKα (6,7). Importantly, the interaction of RAF1 with ROKα is promoted by RAF1/BRAF heterodimerization and the resulting ERK activation. Thus, RAF1 is a paradigm signaling hub precisely coordinating the proper timing of ERK-induced proliferation with ROKα-induced changes in cell shape, adhesion, or motility (8).

Similarly, downstream of RAF, MEK1 ensures the temporal coordination of ERK activation with the termination of the PI3-Kinase signal. Direct negative feedback phosphorylation of MEK1 by ERK is necessary for the correct localization of the phosphatase PTEN to the membrane, where PTEN limits PIP3 accumulation and thus shuts down the parallel PI3 Kinase/AKT pathway (9). By limiting AKT/mTORC1 activation the feedback phosphorylation of MEK1 regulates the return of activated hematopoietic stem cells (HSC) to quiescence and prevents HSC exhaustion during stress hematopoiesis (10).

Thus, both RAF1 and MEK1 are moonlighting proteins, which have evolved to retain their ancestral function (phosphorylation of their targets) and their place in the pathway but have acquired further, essential roles which cannot be compensated by other paralogs.

Expected and unexpected functions in tumorigenesis and inflammation

BRAF and RAF1 are essential for the development and maintenance of RAS-driven epidermal tumors (11) and for tumor angiogenesis (12). In this, as in other contexts, the two paralogs have distinct essential roles. BRAF acts as a MEK/ERK activator promoting proliferation and angiogenesis (12). In contrast, RAF1 works as an endogenous ROKα inhibitor essential to prevent keratinocyte differentiation (13) and to promote the collective migration of endothelial cells by strengthening cell-cell adhesions (14). Compound ablation of both RAF paralogs in the epidermis causes flash regression of RAS-driven tumors; the long term effect of this, however, is the development of a severe inflammatory allergic disease with the hallmarks of human atopic dermatitis (15).

Finally, although essential for the development of RAS-driven skin and lung tumors (11,15,16,17), RAF1 surprisingly features as a negative regulator of liver cancer. The phenotype is ERK-independent, and relies on the increased expression of the known liver oncogene YAP1 and on the phosphorylation of the transcription factor STAT3. In contrast, RAF1 is required in macrophages to create an environment which stimulates growth of hepatocellular carcinoma (18). Mechanistic insight into the multifaceted functions of RAF1 in different cell types, tissues and in tumors is key for a better understanding of RAF1 in tumorigenesis and for the development of future therapeutic interventions.

The lab continues to focus on deciphering signaling pathways in the context of the whole organism, using a blend of genetics, molecular biology, biochemistry and cell biology.

Manuela Baccarini is the coordinator of the PhD programme “Signaling mechanism in cellular homeostasis” (W1261), and of the Vienna Doctoral School "Molecules of Life" with the mission to educate excellent PhD students to become independent researchers with a competitive professional profile by fostering independence, inquisitive thinking and scientific rigor.

References

1. Dorard C, Vucak G, Baccarini M. Deciphering the RAS/ERK pathway in vivo. Biochem. Soc. Trans. 45(1):27-36 (2017) 

2. Desideri E, Cavallo AL, Baccarini M. Alike but Different: RAF Paralogs and Their Signaling Outputs. Cell 161(5):967-70 (2015) 

3. Matallanas, D. et al. RASSF1A elicits apoptosis through an MST2 pathway directing proapoptotic transcription by the p73 tumor suppressor protein. Mol Cell 27, 962-75 (2007). 

4. O'Neill E, Rushworth L, Baccarini M & Kolch W. Role of the kinase MST2 in suppression of apoptosis by the proto-oncogene product Raf-1. Science 306, 2267-70 (2004). 

5. Yamaguchi O, et al. Cardiac-specific disruption of the c-raf-1 gene induces cardiac dysfunction and apoptosis. J Clin Invest 114, 937-43 (2004). 

6. Ehrenreiter K, et al. Raf-1 regulates Rho signaling and cell migration. J Cell Biol 168, 955-64 (2005). 

7. Piazzolla D, Meissl K, Kucerova L, Rubiolo C & Baccarini M. Raf-1 sets the threshold of Fas sensitivity by modulating Rok-{alpha} signaling. J. Cell Biol. 171, 1013-1022 (2005). 

8. Varga A, Ehrenreiter K, Aschenbrenner B, Kocieniewski P, Kochanczyk M, Lipniacki T, Baccarini M. RAF1/BRAF dimerization integrates the signal from RAS to ERK and ROKα. Sci Signal 10, 469 (2017) 

9. Zmajkovicova K, Jesenberger V, Catalanotti F, Baumgartner C, Reyes G, Baccarini M. MEK1 is required for PTEN membrane recruitment, AKT regulation, and the maintenance of peripheral tolerance. Mol Cell 50(1):43-55 (2013) 

10. Baumgartner C, Toifl S, Farlik M, Halbritter F, Scheicher R, Fischer I, Sexl, V, Bock C, Baccarini M. An ERK-dependent feedback mechanism prevents hematopoitetic stem cell exhaustion. Cell Stem Cell.;22(6):879-892 (2018)

10. Kern F, Doma E, Rupp C, Niault T & Baccarini M. Essential, non-redundant roles of B-Raf and Raf-1 in Ras-driven skin tumorigenesis. Oncogene, 32, 2483-92 (2013) 

11. Sobczak I, et al. B-Raf is required for ERK activation and tumor progression in a mouse model of pancreatic beta-cell carcinogenesis. Oncogene 27, 4779-87 (2008). 

12. Niault, T, Sobczak, I., Meissl, K., Weitsman, G., Piazzolla, D., Maurer, G., Kern, F., Ehrenreiter, K., Hamerl, M., Moarefi, I., Leung, T., Carugo, O., Ng, T., and Baccarini, M. From autoinhibition to inhibition in trans: the Raf-1 regulatory domain inhibits Rok-alpha kinase activity. J Cell Biol, 187, 335-342 (2009) 

13. Wimmer R, Cseh B, Maier B, Scherrer K & Baccarini M. Angiogenic sprouting requires the fine tuning of endothelial cell cohesion by the Raf-1/Rok-alpha complex. Developmental cell, 22, 158-171, (2012) 

13. Ehrenreiter K et al. Raf-1 addiction in Ras-induced skin carcinogenesis. Cancer Cell 16, 149–160 (2009). 

14. Raguz J, et al. Epidermal RAF prevents allergic skin disease. eLife; 19;5. pii: e14012. (2016)  

15. Karreth FA, Frese KK, Denicola GM, Baccarini M & Tuveson DA. C-Raf is required for the initiation of lung cancer by K-Ras. Cancer Discov. 1, 128–136 (2011) 

16. Blasco R. B et al. c-Raf, but not B-Raf, Is essential for development of K-Ras oncogene-driven non-small cell lung carcinoma. Cancer Cell 19, 652–663 (2011) 

17. Jeric I, et al. A cell-autonomous tumour suppressor role of RAF1 in hepatocarcinogenesis. Nat Commun. 21;7:13781 (2016)