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Antibacterial peptides of the innate immune system combat pathogenic microbes, but often have additional roles in promoting inflammation and as growth factors during tissue repair. Midkine (MK) and pleiotrophin (PTN) are the only two members of a family of heparin-binding growth factors. They show restricted expression during embryogenesis and are up-regulated in neoplasia. In addition, MK shows constitutive and inflammation-dependent expression in some non-transformed tissues of the adult. In the present study, we show that both MK and PTN display strong antibacterial activity, present at physiological salt concentrations. Electron microscopy of bacteria and experiments using artificial lipid bilayers suggest that MK and PTN exert their antibacterial action via a membrane disruption mechanism. The predicted structure of PTN, employing the previously solved MK structure as a template, indicates that both molecules consist of two domains, each containing three antiparallel β-sheets. The antibacterial activity was mapped to the unordered C-terminal tails of both molecules and the last β-sheets of the N-terminals. Analysis of the highly conserved MK and PTN orthologues from the amphibian Xenopus laevis and the fish Danio rerio suggests that they also harbor antibacterial activity in the corresponding domains. In support of an evolutionary conserved function it was found that the more distant orthologue, insect Miple2 from Drosophila melanogaster, also displays strong antibacterial activity. Taken together, the findings suggest that MK and PTN, in addition to their earlier described activities, may have previously unrealized important roles as innate antibiotics.

The mammalian focal adhesion kinase (FAK) family of nonreceptor protein-tyrosine kinases have been implicated in controlling a multitude of cellular responses to the engagement of cell surface integrins and G protein-coupled receptors. We describe here aDrosophila melanogaster FAK homologue, DFak56, which maps to band 56D on the right arm of the second chromosome. Full-length DFak56 cDNA encodes a phosphoprotein of 140 kDa, which shares strong sequence similarity not only with mammalian p125FAKbut also with the more recently described mammalian Pyk2 (also known as CAKβ, RAFTK, FAK2, and CADTK) FAK family member. DFak56 has intrinsic tyrosine kinase activity and is phosphorylated on tyrosine in vivo. As is the case for FAK, tyrosine phosphorylation of DFak56 is increased upon plating Drosophila embryo cells on extracellular matrix proteins. In situ hybridization and immunofluorescence staining analysis showed that DFak56 is ubiquitously expressed with particularly high levels within the developing central nervous system. We utilized the UAS-GAL4 expression system to express DFak56 and analyze its function in vivo. Overexpression of DFak56 in the wing imaginal disc results in wing blistering in adults, a phenotype also observed with both position-specific integrin loss of function and position-specific integrin overexpression. Our results imply a role for DFak56 in adhesion-dependent signaling pathways in vivo during D. melanogasterdevelopment.

PRK1 (PKN) is a serine/threonine kinase that has been shown to be activated by RhoA (Amano, M., Mukai, H., Ono, Y., Chihara, K., Matsui, T., Hamajima, Y., Okawa, K., Iwamatsu, A., and Kaibuchi, K. (1996) Science 271, 648–650). Detailed analysis of the PRK1 region involved in RhoA binding has revealed that two homologous sequences within the HR1 domain (HR1a and HR1b) both bind to RhoA; the third repeat within this domain, HR1cPRK1, does not bind RhoA. The related HR1 motif is also found to confer RhoA binding activity to the only other fully cloned member of this kinase family, PRK2. Furthermore, the predictive value of this motif is established for an HR1a sequence derived from aCaenorhabditis elegans open reading frame encoding a protein kinase of unknown function. Interestingly, the HR1aPRK1 and HR1bPRK1 subdomains are shown to display a distinctive nucleotide dependence for RhoA binding. HRIaPRK1 is entirely GTP-dependent, while HR1bPRK1 binds both GTP- and GDP-bound forms of RhoA. This distinction indicates that there are two sites of contact between RhoA and PRK1, one contact through a region that is conformationally dependent upon the nucleotide-bound state of RhoA and one that is not. Analysis of binding to Rho/Rac chimera provides evidence for a HR1aPRK1 but not HR1bPRK1 interaction in the central third of Rho. Additionally, it is observed that the V14RhoA mutant binds HR1a but does not bind HR1b. This distinct binding behavior corroborates the conclusion that there are independent contacts on RhoA for the HR1aPRK1 and HR1bPRK1motifs.

As potential targets for polyphosphoinositides, activation of protein kinase C (PKC) isotypes (beta 1, epsilon, zeta, nu) and a member of the PKC-related kinase (PRK) family, PRK1, has been compared in vitro. PRK1 is shown to be activated by both phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P2) as well as phosphatidylinositol 3,4,5-trisphosphate (PtdIns-3,4,5-P3) either as pure sonicated lipids or in detergent mixed micelles. When presented as sonicated lipids, PtdIns-4,5-P2 and PtdIns-3,4,5-P3 were equipotent in activating PRK1, and, furthermore, sonicated phosphatidylinositol (PtdIns) and phosphatidylserine (PtdSer) were equally effective. In detergent mixed micelles, PtdIns-4,5-P2 and PtdIns-3,4,5-P3 also showed a similar potency, but PtdIns and PtdSer were 10-fold less effective in this assay. Similarly, PKC-beta 1, -epsilon, and -nu were all activated by PtdIns-4,5-P2 and PtdIns-3,4,5-P3 in detergent mixed micelles. The activation constants for PtdIns-4,5-P2 and PtdIns-3,4,5-P3 were essentially the same for all the kinases tested, implying no specificity in this in vitro analysis. Consistent with this conclusion, the effects of PtdIns-4,5-P2 and PtdIns-3,4,5-P3 were found to be inhibited at 10 mM Mg2+ and mimicked by high concentrations of inositol hexaphosphate and inositol hexasulfate. The similar responses of these two classes of lipid-activated protein kinase to these phosphoinositides are discussed in light of their potential roles as second messengers.