Dictyostelium discoideum possesses more EGF-like (EGFL) domains than any other sequenced eukaryote. Here we show that a synthetic EGFL peptide (DdEGFL1) based upon an amino acid sequence from a cysteine-rich Dictyostelium protein, functions extracellularly to enhance random cell motility and cAMP-mediated chemotaxis in Dictyostelium by 625% and 85%, respectively, in strain NC4 and by 620% and 80% in strain AX3. Quinacrine inhibited peptide-enhanced random motility but not chemotaxis in strain AX3 providing evidence that PLA2 is the predominant regulator of this process. While LY294002 alone had no significant effect on either event, in combination with quinacrine it dramatically inhibited both processes suggesting that both PI3K and PLA2-mediated signaling are required for EGFL peptide-enhanced cell movement. DdEGFL1 also sustained the threonine phosphorylation of a 210kDa protein that is dephosphorylated during Dictyostelium starvation. Taken together, these results suggest an important role for certain EGFL peptides in Dictyostelium cell movement.

Probing of a cDNA expression library from multicellular development of Dictyostelium discoideum using a recombinant radiolabelled calmodulin probe (35S-VU1-CaM) led to the isolation of a cDNA encoding a putative CaM-binding protein (CaMBP). The cDNA contained an open reading frame of 951 bp encoding a 227aa polypeptide (25.5 kDa). Sequence comparisons led to highly significant matches with cytosolic thymidine kinases (TK1; EC 2.7.1.21) from a diverse number of species including humans (7e-56; 59% Identities; 75% Positives) indicating that the encoded protein is D. discoideum TK1 (DdTK1; ThyB). DdTK1 has not been previously characterized in this organism. In keeping with its sequence similarity with DdTK1, antibodies against humanTK1 recognize DdTK1, which is expressed during growth but decreases in amount after starvation. A CaM-binding domain (CaMBD; 20GKTTELIRRIKRFNFANKKC30) was identified and wild type DdTK1 plus two constructs (DdTK deltaC36, DdTK deltaC75) possessing the domain were shown to bind CaM in vitro but only in the presence of calcium while a construct (DdTK deltaN72) lacking the region failed to bind to CaM. Thus, DdTK1 is a Ca2+-dependent CaMBP. Sequence alignments against TK1 from vertebrates to viruses show that CaM-binding region is highly conserved. The identified CaMBD overlaps the ATP-binding (P-loop) domain suggesting CaM might affect the activity of this kinase. Recombinant DdTK is enzymatically active and showed stimulation by CaM (113+/-0.5%) an in vitro enhancement that was prevented by co-addition of the CaM antagonists W7 (91.2+/-0.8%) and W13 (96.6+/-0.6%). The discovery that TK1 from D. discoideum, and possibly other species including humans and a large number of human viruses, is a Ca2+-dependent CaMBP opens up new avenues for research on this medically relevant protein.