Functional Genomics Approach to Identify Targets of MPK-1 ERK Signaling
in Germline Development
Swathi Arur1, Mitsue Ohmachi1, Sudhir Nayak1, Tina Chang1, Andy Golden2, Tim
Schedl1
11Dept of Genetics, Washington University School of Medicine, St. Louis MO,
2National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda
MD
RAS/ERK signaling is important for many aspects of normal development while
inappropriate signaling can predispose to cancer. In C. elegans MPK-1 ERK is critical for
spatial co-ordination of meiotic prophase progression and oogenesis and regulates nine
different germline processes. The cellular response to signaling occurs as a consequence
of ERK activating or inactivating target proteins by phosphorylation. However, targets
of MPK-1 that mediate germline development are unknown. To identify downstream
targets of MPK-1 signaling, we are using an integrated in silico, reverse genetic and
biochemical approach.
In silico searches identified >2000 proteins from the C. elegans proteome that
contain putative ERK docking (e.g. D-domain) and phospho-acceptor (S/TP) sites. 156
were chosen for RNAi screening based on a) multiple docking sites, b) conservation
of docking sites and c) germline expression. MPK-1 regulates a number of germline
processes and for execution of an individual process it likely phosphorylates multiple
targets. Thus RNAi based gene inactivation of each target is performed in sensitized
genetic backgrounds to permit detection of small or partial changes in the pathway that
controls a given cellular process.
To date, we have identified 34 genes that behave genetically as mpk-1 targets for
germline development. RNAi of 25 genes enhances specific phenotypes of an mpk-1
temperature sensitive (ts) loss-of-function mutant while RNAi of 9 genes enhance let-60
RAS ts gain-of-function. Biochemical analysis has thus far shown that 11 of 13 tested
gene products identified by RNAi are direct in vitro substrates of activated mammalian
ERK2. Together, this work has identified at least 11 gene products representing diverse
functional categories that are likely direct targets that execute MPK-1 ERK regulated
aspects of germline development. Given the conservation of the targets and their docking
sites it is likely that the mouse/human orthologous proteins are previously unknown
targets of ERK phosphorylation.