MPK-1 signaling controls seven biological processes during C. elegans germ-line development. (A) Schematic of the conserved RAS–ERK signaling pathway in mammals and C. elegans. ERK/MPK-1 activates or inactivates substrate proteins via phosphorylation. (B) MPK-1 controls seven biological processes during germ-line development (11). Fluorescent micrograph of a dissected C. elegans adult hermaphrodite gonad showing chromosome (blue), activated dpMPK-1 (red), and membrane (green) morphology as germ cells progress through oogenesis. The seven MPK-1 controlled germ-line processes examined in this study are indicated (11) (Fig. S1).

In silico and genetic enhancer screen identifies 37 candidate MPK-1 substrates that function during germ-line development. (A) Outline of bioinformatics approach and sequences of characterized ERK-docking sites (DS). (B) Genetic logic used in the enhancer screen to identify genes that promote or inhibit MPK-1 pathway function. Wild-type control and sensitized backgrounds contain the rrf-1(pk1417) mutation, which prevents RNAi in the soma but not the germ line (29). At the permissive temperature where the RNAi screen was performed, mpk-1(ga111ts) ERK has lower MPK-1 activation whereas let-60(ga89ts) RAS has elevated/ectopic MPK-1 activation. (C) Pie chart showing predicted molecular functions of 37 genes that enhance one or the other sensitized genetic background (Dataset S2). (D) Fluorescent micrographs illustrating ddx-19 RNAi enhancement of rrf-1; mpk-1(ga111ts). (E) Fluorescent micrographs illustrating gsk-3 attenuated RNAi enhancement of rrf-1;let-60(ga89ts). (Scale bar, 20 μm.)

Multiple molecularly diverse substrates function in each MPK-1-dependent process. (A) (Top) Seven MPK-1-dependent processes, shown relative to adult hermaphrodite germ-line development. (Middle and Bottom) Thirty in vitro ERK2 substrates and their functional molecular categories. +, Germ-line process in which a given substrate functions. Analysis with null alleles for 10 substrates (underlined) (Dataset S2), reveals concordant results with RNAi screen. For oocyte growth control, two substrates enhance mpk-1(ga111ts) and produce large oocytes whereas eight enhance let-60(ga89ts) and produce small oocytes (Fig. S1). (B) Nonoverlapping groups of MPK-1 substrates execute pachytene cellular organization (red circle, 7 genes) and germ cell apoptosis (green circle, 4 genes). (C) Partially overlapping groups of substrates regulate pachytene cellular organization (red) and/or oocyte organization and differentiation (blue).

DDX-19 and GSK-3 phosphorylation is MPK-1-dependent in vivo. (A and D) ClustalW protein alignment of C. elegans and human orthologs of DDX-19 (A) identifies two conserved phosphoacceptors (S612 and T745; red circles) and two conserved docking sites (red boxes) and GSK-3 (D) identifies four conserved phosphoacceptor sites (T254, T289, T304, and T310; red circles) and two conserved docking sites (one shown here, red boxes) (Fig. S4). Mapping of ERK2 phosphoacceptors for (B) DDX-19 and (E) GSK-3 using in vitro kinase assays and site-directed mutagenesis (SI Methods). (B and D) (Top) ³²P incorporation into indicated proteins. (Middle) Western blot analysis by using anti-HIS antibody for loading control. (Bottom) Kinetic analyses were performed on wild-type and all phosphoacceptor mutant proteins (S/T to A). Values for K_m and V_max (average ± 1 SD of five experiments) were used to calculate the RAR, which was normalized to 1, relative to MBP. LIN-1 is a positive control. (C and F) Western blot analysis of protein extracts from wild-type (WT), mpk-1(ga117)-null, and (C) ddx-19(ok783)-null or (F) gsk-3(nr2047)-null animals probed with antibodies specific for (C) phospho-DDX-19 (pS612 and pT745), total DDX-19, and paramyosin (loading control), (F) phospho-GSK-3 (pT304 and pT310), total GSK-3, and α-tubulin (loading control). The antibodies are specific to the gene product tested because no species was detected in the respective null mutant. DDX-19 is phosphorylated on S612 and/or T745 in WT but not mpk-1 null. *, slower-mobility GSK-3 band detected with total GSK-3 antibody that comigrates with the species detected with the phospho-GSK-3 antibody. In mpk-1-null, this band is not detected on short exposures; however, on longer exposures, a weak band is visible (also weakly detected by total-GSK-3 antibody).

MPK-1 substrates function to regulate MPK-1 activation in feedback loops. (A) Ten MPK-1 substrates regulate MPK-1 activation and fall into three categories. (B) Fluorescent micrographs of dissected rrf-1 null gonads, after either gfp RNAi (Upper) or rba-1 RNAi (Lower), stained for dpMPK-1 (red) or DNA (blue). (Scale bar, 20 μm.) (C) Models of possible regulatory feedback loops through which substrates modulate germ-line MPK-1 activation.