SDF-2 induction of terminal differentiation in Dictyostelium discoideum ismediated by the membrane-spanning sensor kinase DhkA

SDF-2 is a peptide released by prestalk cells during culmination that stimu lates prespore cells to encapsulate. Genetic evidence indicates that the re sponse is dependent on the dhkA gene. This gene encodes a member of the his tidine kinase family of genes that functions in two-component signal transd uction pathways. The sequence of the N-terminal half of DhkA predicts two h ydrophobic domains separated by a 310-amino-acid loop that could bind a lig and. By inserting MYC, epitopes into DhkA, we were able to show that the lo op is extracellular while the catalytic domain is cytoplasmic. Cells expres sing the MYC epitope in the extracellular domain of DhkA were found to resp ond only if induced with 100-fold-higher levels of SDF-2 than required to i nduce dhkA(+) cells; however, they could be induced to sporulate by additio n of antibodies specific to the MYC epitope. To examine the enzymatic activ ity of DhkA, we purified the catalytic domain following expression in bacte ria and observed incorporation of labelled phosphate from ATP consistent wi th histidine autophosphorylation. Site-directed mutagenesis of histidine(13 95) to glutamine in the catalytic domain blocked autophosphorylation. Furth ermore, genetic analyses showed that histidine(1395) and the relay aspartat e(2075) of DhkA are both critical to its function but that another histidin e kinase, DhkB, can partially compensate for the lack of DhkA activity. Spo rulation is drastically reduced in double mutants lacking both DhkA and Dhk B. Suppressor studies indicate that the cyclic AMP (cAMP) phosphodiesterase RegA and the cAMP-dependent protein kinase PKA act downstream of DhkA.