Protein Kinase D (PKD) isoforms are Ser/Thr protein kinases that mediate signal transduction downstream from phospholipase C and diacylglycerol (DAG). Little is known about in vivo regulation, downstream effectors and physiological functions of PKDs in normal differentiated cells. The
dkf-2 (D-Kinase Family-2) gene encodes two C. elegans PKDs, DKF-2A and DKF-2B. DKF-2A is selectively and robustly expressed in intestine under control of GATA enhancer elements. Microarray analysis (22,500 transcripts per chip) was used to determine if DKF-2A regulates gut-specific gene expression. Overexpression of DKF-2A caused 3-40 fold inductions of >50 mRNAs that encode proteins involved in protecting C. elegans against invading pathogenic bacteria (innate immune response). Included are antimicrobial peptides (caenacins, C-lectins, abf, saposins), proteases, detoxifying enzymes and transporters. qRT-PCR analysis confirmed the microarray data and showed the same genes are up-regulated when C. elegans gut is invaded by P. aeruginosa (PA14). Animals carrying a disrupted
dkf-2 gene are viable and reproduce normally. Thus, DKF-2 depleted worms and
dkf-2 null animals reconstituted with 3X DKF-2A-GFP were fed PA14 pathogen. DKF-2 deficient animals are hypersensitive to infection and killing; only 18% remained alive at 62h, whereas 50% of WT animals survived at 62h. DKF-2A overexpression conferred enhanced resistance to PA14 (79% survivors, 62h). Detailed survival curves confirmed these results. Thus, DKF-2A links DAG-mediated signal transduction to regulation of innate immunity. Expression of DKF-2A catalytic activity depends on upstream activators that promote phosphorylation of 2 serines in the activation loop of the D kinase. Deficiency of essential upstream signaling proteins will block phosphorylation/activation of DKF-2A, thereby mimicking the
dkf-2 null phenotype in the presence of excess DKF-2A. C. elegans expressing excess DKF-2A, but lacking TPA-1 have essentially the same hypersensitivity to PA14 as
dkf-2 null animals. Thus, TPA-1, which is homologous with mammalian PKC delta, is an indispensable upstream regulator of DKF-2A activity and anti-microbial functions in intestinal cells in vivo. A series of preliminary crosses also indicates that the DAG-TPA-1-DKF-2A pathway intersects with the
nsy-1—
sek-1—
pmk-1 pathway at an upstream location. In summary, DKF-2A links DAG-mediated signal transduction to regulation of gene expression that protects C. elegans against pathogenic bacteria. TPA-1 is a critical regulator of DKF-2A; DKF-2A controls inducible expression of anti-microbial proteins.