SEL-10/Fbw7-dependent negative feedback regulation of LIN-45/Braf signaling in C. elegans via a conserved phosphodegron

Abstract

The conserved E3 ubiquitin ligase component named SEL-10 in Caenorhabditis elegans and Fbw7 in mammals targets substrates for ubiquitin-mediated degradation through a high-affinity binding site called a Cdc4 phosphodegron (CPD). As many known substrates of Fbw7 are oncoproteins, the identification of new substrates may offer insight into cancer biology as well as aspects of proteome regulation. Here, we evaluated whether the presence of an evolutionarily conserved CPD would be a feasible complement to proteomics-based approaches for identifying new potential substrates. For functional assessments, we focused on LIN-45, a component of the signal transduction pathway underlying vulval induction and the ortholog of human Braf, an effector of Ras in numerous cancers. Our analysis demonstrates that LIN-45 behaves as a bona fide substrate of SEL-10, with mutation of the CPD or loss of sel-10 resulting in increased activity and protein stability in vivo. Furthermore, during vulval induction, the downstream kinase MPK-1/ERK is also required for LIN-45 protein degradation in a negative feedback loop, resulting in degradation of LIN-45 where ERK is highly active. As the CPD consensus sequence is conserved in human Braf, we propose that Fbw7 may also regulate Braf stability in some cell contexts. We discuss the implications of our findings for vulval development in C. elegans, the potential applicability to human Braf, and the value of a CPD-based predictive approach for human Fbw7 substrates.

Figure 1.

C. elegans LIN-45 has a conserved CPD and is negatively regulated by SEL-10. (A) Alignment of high-affinity CPD motifs from validated human substrates and C. elegans LIN-12. Residues essential for Fbw7 binding or Fbw7-mediated degradation are shown in red here and in B. As mutations in the “central threonine” and “+4 serine” residues were incorporated into LIN-45, we note that in all cases shown here, mutation of these residues to alanine has been documented to have one or more of the following consequences: stabilization of the substrate, increased activity of the substrate, and/or loss of Fbw7 binding to the substrate (for LIN-12, see Supplemental Fig. 1). (B) Potential conserved high-affinity CPDs identified in C. elegans LIN-45 and human Braf. Conserved Raf functional domains (black) include the Ras-binding and cysteine-rich domains (RB), 14-3-3 binding domain, and kinase domain. LIN-45 has a single predicted high-affinity CPD (black arrow; residues 431–436 of C. elegans), conserved in three other nematode species analyzed. Braf has a predicted high-affinity CPD (black arrow; residues 400–405) in the corresponding position, conserved in zebrafish (and other vertebrates). Human Braf also has a second predicted CPD (gray arrow; residues 332–337 of human) that did not fulfill our conservation criterion. Full alignments of the region between the Ras-binding and kinase domains are shown in Supplemental Figure 2. (C) sel-10(0) suppresses lin-45 partial loss of function. Rod-like larval lethality (Let) associated with lin-45(h) hypomorphic alleles n2506 and n2018 and suppression by the null sel-10(ar41) as percentage of total progeny. (*) P-value < 0.0001 for comparisons of lin-45 to lin-45; sel-10 strains. The number of larvae scored (n) is indicated at right. The markers unc-24(e138) and him-5(e1490) were also homozygous in strains containing or compared with lin-45(n2506). The markers dpy-20(e1282) and him-5(e1490) were also homozygous in strains containing or compared with lin-45(n2018). (D) sel-10(0) does not bypass the need for lin-45. The sterile (Ste) and vulvaless (Vul) phenotype of the null allele lin-45(dx19) in sel-10(+) or sel-10(0) as percentage of total adults. Number of adults scored (n) is indicated at right. All animals scored were also homozygous for lon-3(e2175). lin-45(dx19) genotypes were F1 segregrants from lin-45(dx19)/DnT1; lon-3(e2175)/DnT1 or lin-45(dx19)/DnT1; lon-3(e2175) sel-10(ar41)/DnT1 mothers.

Figure 2.

Activated LIN-45 is negatively regulated by SEL-10 and the LIN-45 CPD. (A) Normal pattern of VPC fates. The VPCs (P3.p–P8.p) are initially equivalent. In the L3 stage, the EGF-like inductive signal produced by the anchor cell (AC) of the gonad results in high EGFR–Ras–Raf–ERK activity, causing P6.p to adopt the 1° vulval fate and produce lateral signal ligands such as LAG-2 (for review, see Sternberg 2005). This lateral signal results in high LIN-12/Notch activity in P5.p and P7.p, which adopt the 2° vulval fate. P3.p, P4.p, and P8.p have the potential to adopt vulval fates but do not because they lie outside the range of the spatial patterning signals. (B) Photomicrographs of the hermaphrodite mid-body. (Top) A wild-type hermaphrodite has a normal vulva (*). (Bottom) Constitutive Ras–Raf–ERK activity causes outer VPCs to adopt vulval fates and produce pseudovulvae (arrows), resulting in a multivulva phenotype, as shown in a representative sel-10(ar41); arEx1292[lin-45(ED)] hermaphrodite. (C) sel-10(0) enhances lin-45 constitutive activity but not lin-45(+). The penetrance and expressivity of the multivulva phenotype caused by constitutively active forms of LIN-45 [LIN-45(ED) or LIN-45(V627E)] is enhanced by sel-10(ar41). Shown here is the percentage of adult hermaphrodites carrying the transgenes arEx1300[lin-45(+)], arEx1474 [lin-45(V627E)], or arEx1292[lin-45(ED)] displaying one, two, or three or more large ectopic pseudovulvae. All strains also were homozygous for him-5(e1490). (*) P-value < 0.0001 for comparisons of control and sel-10 strains. The number of adults scored (n) is indicated at right. (D) sel-10(0) enhances the ectopic 1° fate specification caused by LIN-45(ED). The percentage of L3 hermaphrodites carrying arEx1292[lin-45(ED)] that display ectopic expression of the reporter arIs131[lag-2p∷2xnls∷yfp] in Pn.px and Pn.pxx stage cells. Ectopic expression of lag-2p∷yfp was categorized as “alternating 1°” when VPCs adjacent to the ectopic expression were lag-2p∷yfp-negative and as “adjacent 1°” when the neighboring VPCs were lag-2p∷yfp-positive. When two adjacent VPCs adopt the 1° fate, Raf activity is not only high enough to induce a vulval fate, it must be sufficiently high to override LIN-12/Notch activity, which acts to oppose Ras–Raf–ERK activity (Sternberg 2005). Both strains also were homozygous for him-5(e1490). (*) P-value < 0.0001 for comparisons of all ectopic 1° fate as well as adjacent 1° fate observed in control and sel-10 strains. The number of L3 larvae scored (n) is indicated at right. (E) CPD mutations enhance lin-45 constitutive activity but not lin-45(+). The penetrance and expressivity of the multivulva phenotype caused by constitutively active forms of LIN-45 [LIN-45(ED) or LIN-45(V627E)] is enhanced by mutating the central threonine and +4 serine of the CPD to alanine; i.e., T432A and S436A (AA). Shown here is the percentage of adult hermaphrodites carrying transgenes expressing the indicated forms of LIN-45 displaying one, two, or three or more large ectopic pseudovulvae. Individual bars represent independent strains isolated for each transgene. Transgenes scored were arEx1300 and arEx1301 for (+); arEx1294 and arEx1296for (AA); arEx1474, arEx1475, and arEx1476 for (V627E); arEx1479, arEx1483, and arEx1485 for (AA,V627E); arEx1291, arEx1292, and arEx1335 for (ED); and arEx1287, arEx1288, and arEx1337 for (AAED). The number of adults scored (n) is indicated at right.

Figure 3.

LIN-45 is down-regulated post-translationally in P6.p, the VPC where Ras–Raf–ERK signaling is activated. (A) Normal pattern of P5.p, P6.p, and P7.p fates and LIN-45/Raf activity. Ras–Raf–ERK activity is high in P6.p, which adopts the 1° vulval fate, and low in other VPCs, including P5.p and P7.p, which adopt the 2° vulval fate. After fate specification, VPCs divide, and the daughter “Pn.px” cells are fully committed. Except when noted, all quantification for lin-45 reporters was done by scoring hermaphrodites at Pn.px and Pn.pxx stages to ensure that the specification process has been completed. (B) The fosmid[yfp-lin-45] reporter. (Top) Photomicrograph of YFP-LIN-45 expressed from arEx1482 in VPCs. (AC) Anchor cell of the gonad. (Bottom) YFP-LIN-45 in a Pn.px stage hermaphrodite, showing that YFP-LIN-45 has been down-regulated in the daughters of P6.p but is evident in neighboring cells. (C) The lin-31p∷yfp-lin-45 tissue-specific reporter. To assess post-translational LIN-45 stability, YFP-LIN-45 was expressed in VPCs and their daughters using heterologous regulatory sequences, the lin-31 promoter, and the unc-54 3′ UTR. Shown here is YFP-LIN-45 accumulation in a Pn.px stage hermaphrodite carrying arEx1528. YFP-LIN-45 accumulation was scored for three independent transgenes. (D) The lin-31p∷2xnls-yfp control reporter. The lin-31 promoter and the unc-54 3′ UTR used in C results in uniform expression of 2xNLS-YFP (open arrow). Shown here is 2xNLS-YFP in a Pn.px stage hermaphrodite carrying arEx1541[lin-31p∷2xnls-yfp]. 2xNLS-YFP accumulation was scored for three independent transgenes.

Figure 4.

sel-10 in trans and the CPD in cis are required for down-regulation of LIN-45. (A) YFP-LIN-45 expressed from a fosmid reporter is stabilized in P6.p in a sel-10(0) mutant. (Top) Photomicrograph of YFP-LIN-45 accumulation in a sel-10(ar41); arEx1482[fosmid∷yfp-lin-45] Pn.px stage hermaphrodite. (Bottom) YFP-LIN-45 accumulation was scored for arEx1482 in sel-10(+) and sel-10(0) backgrounds. Both strains were also homozygous for him-5(e1490). (*) P-value < 0.0001 for comparison of P6.p descendants in control and sel-10 strains. (B) YFP-LIN-45 expressed from a tissue-specific reporter is stabilized post-translationally in a sel-10(0) mutant. (Top) Photomicrograph showing YFP-LIN-45 stabilization in a sel-10(ar41); arEx1528[lin-31p∷yfp-lin-45] Pn.px stage hermaphrodite. (Bottom) YFP-LIN-45 accumulation was scored for arEx1528 in sel-10(+) and sel-10(0) backgrounds. Both strains also were homozygous for him-5(e1490). (*) P-value <0.0001 for comparison of P6.p descendants in control and sel-10 strains. (C) The control reporter lin-31p∷2xnls-yfp is uniformly expressed in a sel-10(0) mutant. These data show that the heterologous regulatory sequences are not affected by the sel-10 genotype. 2xNLS-YFP was scored for arEx1541[lin-31p∷2xnls-yfp] in sel-10(+) and sel-10(0) backgrounds. Both strains were also homozygous for him-5(e1490). (D) The LIN-45 CPD is required for down-regulation in P6.p. The CPD mutations T432A and S436A (AA) were introduced in the lin-31p∷yfp-lin-45 reporter context. Shown here is a photomicrograph of YFP-LIN-45(AA) in a Pn.px stage arEx1571[lin-31p∷yfp-lin-45(AA)] hermaphrodite. (E) Mutation of the CPD “central threonine” prevents LIN-45 down-regulation. The mutation T432A was introduced into lin-31p∷yfp-lin-45. YFP-LIN-45(T432A) accumulation was scored for three independent transgenes. (F) Mutation of the CPD “+4 serine” prevents LIN-45 down-regulation. The mutation S436A was introduced into lin-31p∷yfp-lin-45. YFP-LIN-45(S436A) accumulation was scored for three independent transgenes.

Figure 5.

mpk-1 activity in trans and the ERK docking site in cis are required for LIN-45 down-regulation. (A) The ERK docking site of the D-domain type in LIN-45. (Left) The CPD (gray box) is adjacent to a sequence that conforms to an ERK docking site of the D-domain type (black box). (Right) Schematic diagrams of the full-length LIN-45 protein and a shorter interval (residues 218–480) that is sufficient for down-regulation in P6.p. (B) LIN-45(218–480) is sufficient for down-regulation in P6.p. YFP-tagged LIN-45(218–480) accumulation was scored for three independent transgenes. (C) The LIN-45 ERK docking site is required for down-regulation in P6.p. Mutations to disrupt ERK binding (L429A and L431A) were introduced in the lin-31p∷yfp-lin-45 reporter. YFP-LIN-45(L429A,L431A) accumulation was scored for three independent transgenes. (D) MPK-1 is required for LIN-45 down-regulation in P6.p. To reduce mpk-1 activity, the temperature-sensitive mutant mpk-1(ga111) was shifted to 25°C at or after the time of vulval induction. To ensure that P6.p had adopted the 1° fate, mpk-1(+) and mpk-1(ts) strains carrying arEx1528[lin-31p∷yfp-lin-45] were examined in the P6.pxx stage (i.e., after two rounds of cell division); if P6.p had not been induced to the 1° fate, it would have divided only once, as is characteristic for the 3° nonvulval fate. YFP-LIN-45 accumulation persists in induced P6.p descendants. Both strains were also homozygous for him-5(e1490). (*) P-value < 0.0001 for comparison of P6.p in control and mpk-1 strains. (E) The reporter lin-31p∷2xnls-yfp is unaffected by mpk-1. 2xNLS-YFP accumulation was scored at the P6.pxx stage for arEx1541[lin-31p∷2xnls-yfp] in mpk-1(+) and mpk-1(ts) strains at 25°C. Both strains also were homozygous for him-5(e1490).

Figure 6.

Model for negative feedback leading to LIN-45 degradation mediated by SEL-10 and MPK-1. In P6.p, LIN-45 is activated in an EGFR and Ras-dependent manner involving Ras binding, dimerization, and phosphorylation of the LIN-45 activation loop. Active LIN-45 leads to activation of the downstream kinase MPK-1/ERK, resulting in phosphorylation of the LIN-45 CPD as well as MPK-1 substrates that promote the 1° cell fate. The “central threonine” CPD residue of LIN-45 has the hallmark of an ERK phospho-acceptor site, while another unknown kinase is expected to phosphorylate the “+4 serine.” The “+4 serine kinase” may but need not be dependent on Ras–Raf–ERK. When phosphorylated at both critical CPD residues, LIN-45 is recognized by SEL-10 for ubiquitination, leading to degradation. We note that dephosphorylation of LIN-45 and mammalian Raf proteins also occurs (Kao et al. 2004; Ritt et al. 2010), so a “recycling” step may be another fate of phosphorylated LIN-45. In other VPCs, such as P5.p and P7.p, LIN-45 and downstream kinase MPK-1 are inactive, so LIN-45 protein is stable.