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. Author manuscript; available in PMC: 2023 Aug 18.
Published in final edited form as: Cell Rep. 2022 Dec 13;41(11):111822. doi: 10.1016/j.celrep.2022.111822

Figure 6. Protein Kinase A (PKA) genetically interacts with alg-1 and alg-1(S642A).

Figure 6.

(A) Schematic of PKA activation. The consensus sequences for PKA substrates are shown on ALG-1 sequence for serine 642 (Top). The basic side chain of arginine (R) (Red) is commonly found at position -2 and -3 of PKA substrates. PKA is a holoenzyme complex (Bottom) formed by two regulatory subunits and two catalytic subunits: KIN-1 and KIN-2 respectively in C. elegans. Activation of GPCRs and downstream synthesis of cyclic adenosine monophosphate (cAMP) by adenylate cyclase leads to the dissociation of the complex which allows the catalytic subunit to phosphorylate its substrates. (B) ALG-1 peptides are phosphorylated in vitro by PKA at serine 642. The graph indicates the incorporation of 32P as measured with a liquid scintillation counter following the incubation of peptides with recombinant PKA. 18 amino-acid peptides of ALG-1 spanning the S642 phosphorylation site or a peptide in which the serine (S) is replaced with an alanine (A) were used. The error bars show the 95% confidence interval from three independent experiments (n=3). (C) Autoradiogram and Coomassie blue stained gels of in vitro PKA kinase reaction. (D) Alae defects caused by kin-2 RNAi are suppressed by the non-phosphorylatable mutant alg-1(S642A). First stage larvae L1 were fed with bacteria expressing double-stranded RNA targeting kin-2 mRNA or a control double-stranded RNA. Young adults staged wild-type animals or alg-1(S642A) mutants were monitored for alae defects. The p-value was measured by Fisher's exact test. The graph is representative of three biological replicates. (E) alg-1(S642A) suppresses kin-2 loss of function mutant Bag of worms phenotype in alg-2(0) animals. The hypomorphic point mutation kin-2(R92C) was edited with CRISPR-Cas9 in alg-2(0) mutant and in the double mutant alg-2(0);alg-1(S642A). The double mutant alg-2(0); kin-2(R92C) animals die at adult stage caused by a defect in egg laying (Egl) leading to embryos hatching inside the worm (Bag of worms). This phenotype was significantly suppressed in animals that cannot be phosphorylated on ALG-1 serine 642. The sample size (n=) used for quantification for each genotype is indicated and the p-value was calculated with Fisher’s exact test.