A rho-binding protein kinase C-like activity is required for the function of protein kinase N in Drosophila development

Abstract

The Rho GTPases interact with multiple downstream effectors to exert their biological functions, which include important roles in tissue morphogenesis during the development of multicellular organisms. Among the Rho effectors are the protein kinase N (PKN) proteins, which are protein kinase C (PKC)-like kinases that bind activated Rho GTPases. The PKN proteins are well conserved evolutionarily, but their biological role in any organism is poorly understood. We previously determined that the single Drosophila ortholog of mammalian PKN proteins, Pkn, is a Rho/Rac-binding kinase essential for Drosophila development. By performing "rescue" studies with various Pkn mutant constructs, we have defined the domains of Pkn required for its role during Drosophila development. These studies suggested that Rho, but not Rac binding is important for Pkn function in development. In addition, we determined that the kinase domain of PKC53E, a PKC family kinase, can functionally substitute for the kinase domain of Pkn during development, thereby exemplifying the evolutionary strategy of "combining" functional domains to produce proteins with distinct biological activities. Interestingly, we also identified a requirement for Pkn in wing morphogenesis, thereby revealing the first postembryonic function for Pkn.

Figure 1.—

Pkn mutations, deletions, and chimeras used in rescue experiments. (A) A diagram showing the structure of Pkn and the Pkn mutations and deletions used in this study. For the Rok-Pkn, Rhot-Pkn, and Pak-Pkn chimeras, the amino terminus of Pkn (up to and including the three ACC domains) was replaced with the Rho-binding domains of Rok or Rhotekin or the Rac-binding domain of Pak. (B) Binding of Pkn, Pkn^G58A mutation, and ΔACC deletions to Rho1 and Rac1. Flag-tagged Pkn and the various deletions were transfected into 293-T cells. Pull-down assays were performed using GTPγS-loaded recombinant GST-Rho1, GST-Rac1, or GST. Pull downs (left) and samples of the lysates (right) were resolved on SDS–PAGE gels, blotted to PVDF, and probed with an anti-flag antibody. (C) Binding of Pkn, the ΔACC123 deletion, and the Pak-Pkn, Rok-Pkn, and Rhotekin-Pkn chimeras to Rho1 and Rac1. Pull-down binding assays were performed as described in B. The pull downs are shown on the left and samples of the lysates are shown on the right.

Figure 2.—

Wing phenotypes generated upon overexpression of the Pkn or PKC53E kinase domain. Transgenic flies carrying the UAS-Pkn kinase domain or UAS-PKC53E kinase domain were crossed to w¹¹¹⁸ flies (A and B) or to the following GAL4 drivers: Act88f (C and D), engrailed (en) (E and F), patched (ptc) (G and H), or Cy6 (I and J), and the wings of the progeny were examined for visible phenotypes. The insets in E and G represent higher-magnification images of areas indicated by the arrows to show the planar polarity defect. The penetrance for each phenotype is indicated as a percentage. The number of flies scored in each case is shown in parentheses. Arrows indicate alterations in wing hair polarity, arrowheads indicate ectopic wing veination, and asterisks indicate increased hair length at the posterior wing margin.

Figure 3.—

PKC53E chimeras used in rescue experiments. (A) A diagram showing the structure of PKC53E and the PKC53E chimeras used in this study. For the Rok-53E, Rhot-53E, and Pak-53E chimeras, the Rho-binding domains of Rok or Rhotekin or the Rac-binding domain of Pak were fused to the amino terminus of PKC53E. For the Pkn-PKC53E chimera, the kinase domain of Pkn was replaced with that of PKC53E. (B) Binding of Pkn, PKC53E, and the Pak-53E, Rok-53E, and Rhot-53E chimeras to Rho1 and Rac1. Pull-down binding assays were performed as described in Figure 1B. The pull downs are shown on the left and samples of the lysates are shown on the right.

Figure 4.—

Wing phenotypes observed in “rescued” flies. (A) Wing from a Pkn^P /+ fly that shows no phenotype. (B) Example of a wing from Pkn^P flies rescued with wild-type Pkn showing ectopic wing veination. (C) Example of a wing from Pkn^P flies rescued with the Pkn-PKC53E chimera showing missing wing veination. (D) Example of a wing from Pkn^P flies rescued with the Pkn-PKC53E chimera showing a reduction in wing size. Ectopic veination is indicated with an arrow. Missing vein material is indicated with an arrowhead.