A depicts locations of C1, PH and kinase domains along the DKF-2 polypeptide. B, animals expressing a dkf-2::DKF-2-GFP transgene were created. GFP-derived fluorescence was recorded using a Zeiss Axio Imager Z1 microscope and AxioVision software. Arrows mark anterior and posterior ends of the intestine. B1 (L2 larva) and B3 (late embryo) show DKF-2-GFP is dispersed in intestinal cells. B2 and B4 are Nomarski interference images of B1 and B3, respectively. C is a Western immunoblot containing cytosolic (S) and membrane (P) proteins (30 μg/lane) isolated from HEK293 cells expressing DKF-2-GFP (lanes 1–4) or DKF-2 (lanes 5–8). Cells were incubated with 0.3 μM PMA (10 min) as indicated. Cytosol and membranes were isolated as previously reported (Feng et al., 2007). The blot was probed with anti-DKF-2 IgGs and chemiluminescence signals were recorded on X-ray film. D, transfected cells expressing DKF-2-GFP or DKF-2 were treated with 0.3 μM PMA or vehicle (basal) for 10 min. Duplicate samples of cells were incubated with pan-PKC inhibitor GF109203X for 1 h prior to PMA addition. Cells were lysed, and DKF-2 proteins were immunoprecipitated and assayed for catalytic activity as described in Experimental Procedures. An immunoblot shows similar amounts of DKF-2 were used in each assay (D, lower panel). Increases in apparent Mr of DKF-2 in PMA-treated cells (C, D) are due to phosphorylation catalyzed by PKCs and other protein kinases.

Microarray analysis measured the abundance of mRNAs in animals expressing an integrated dkf-2::DKF-2-GFP transgene (relative to mRNA levels in WT C. elegans). Animals were fed E. coli OP50. ^& Symbols in parenthesis indicate: (+), mRNA expressed in intestine; (nd), mRNA expression pattern not determined; (−) mRNA expressed in various cells, but not intestine. Designations are based on data provided by WormBase (wormbase.org) and the Genome Sciences Center MultiSAGE C. elegans database (elegans.bcgsc.ca).

A shows induction of mRNAs caused by increased DKF-2 activity or PA14 infection. qRT-PCR data are normalized to actin mRNA and expressed as fold-induction (mRNA concentration in transgenic or infected animals ÷ mRNA concentration in WT animals); nd, not determined. B shows induction of mRNAs in DKF-2 deficient, WT and transgenic (dkf-2::DKF-2-GFP) animals infected with PA14 (20 h). Normalized qRT-PCR data are expressed as fold-induction (mRNA concentration in indicated infected animals ÷ mRNA concentration in uninfected animals). Experiments in A and B were repeated twice, yielding similar results.

A depicts survival curves for dkf-2(pr3) null, pkc-1(ok563);pkc-2(ok328) double null and tpa-1(k530) defective C. elegans mutants feeding on PA14. B shows survival curves for WT, dkf-2(pr3) and transgenic (dkf-2::DKF-2-GFP and tpa-1(k530);dkf-2::DKF-2-GFP) nematodes fed PA14. C presents a model for (partial) regulation of immunity by a DAG→TPA-1→DKF-2 signaling pathway in C. elegans. We speculate that pathogens directly/indirectly activate PLCs, thereby increasing DAG levels. DAG recruits and activates TPA-1, which phosphorylates and activates DAG-bound DKF-2. DKF-2, a PKD prototype, induces expression of immune effector mRNAs that defend intestinal cells against pathogens via PMK-1 dependent and independent mechanisms.

A shows survival curves for WT (N2), dkf-2(pr3) null, transgenic (dkf-2::DKF-2-GFP and dkf-2(pr3);dkf-2::DKF-2-GFP) C. elegans fed with PA14. Assays were replicated 4 times with similar results; typical data are shown. B, proteins (30 μg/lane) from the indicated animals were separated by denaturing electrophoresis and transferred to a PVDF membrane. The blot was probed with anti-DKF-2 IgGs. C shows survival curves for animals fed with E. faecalis OG1RF. Assays were replicated 3 times with similar results. D, WT or transgenic animals were fed with OP50, OP50 HK or PA14 as indicated. Animals were disrupted in a French press, using buffer containing 1% Triton-X100. DKF-2 and DKF2-GFP were immunoprecipitated from samples containing 0.5 mg protein (from equal numbers of animals) by anti-DKF-2 IgGs. Precipitated proteins were assayed for kinase activity.

A, DKF-2 induced 8 categories of effectors that collectively provide an integrated immune response to bacterial infection. B, DKF-2 activation sharply diminished levels of >40 mRNAs.

A shows survival curves for the indicated mutant and transgenic animals upon feeding with PA14. B, amounts of 17 mRNAs were quantified (qRT-PCR) in animals expressing dkf-2::DKF-2-GFP in WT or pmk-1(km25) null backgrounds. Fold-induction is: mRNA concentration in a specified transgenic animal ÷ mRNA concentration in WT C. elegans. C shows effects of DKF-2(A⁹²⁵A⁹²⁹)-GFP and DKF-2(E⁹²⁵E⁹²⁹)-GFP expression on levels of 5 mRNAs in a dkf-2(pr3) background. qRT-PCR data are expressed as fold-induction. D, 30 μg total protein from the indicated WT, null and transgenic strains of C. elegans were assayed by Western immunoblot analysis. The blot was probed with anti-DKF-2 IgGs; DKF-2 polypeptides were detected via chemiluminescence. E, A Western blot (prepared as described in D) was probed with IgGs directed against the di-phosphorylated A-loop of PMK-1 (Promega). Phospho-PMK-1 was detected by chemiluminescence. 14-3-3 protein is a loading control. Experiments in Fig. 6 were performed 3 times, yielding similar results.