Volume 5, Issue 1, Pages 1-11 (January 2000) The Activation Loop of Phosphatidylinositol Phosphate Kinases Determines Signaling Specificity  Jeannette Kunz, Monita P. Wilson, Marina Kisseleva, James H. Hurley, Philip W. Majerus, Richard A. Anderson  Molecular Cell  Volume 5, Issue 1, Pages 1-11 (January 2000) DOI: 10.1016/S1097-2765(00)80398-6

Figure 1 Construction and Analysis of PIP Kinase Activation Loop Chimeras (A) Schematic representation of type I and type II PIP kinase structures indicating the conserved kinase domain, the nonconserved intervening insert region, and the position of the activation loop (AL). (B) Three-dimensional structure of the IIβ monomer. The PtdIns(5)P substrate is modeled into the active site. The endpoints of the activation loop as determined in the three-dimensional structure of IIβ are indicated. (C) Alignment of the activation loop regions of type I, type II, and type III subfamilies of PIP kinases. Alignments were generated by the ClustalW program. The region exchanged between type I and type II PIP kinases is indicated by a black line on top of the alignment. Identical and similar amino acids conserved in all PIP kinases are shaded in dark and light blue, respectively. Amino acid residues within the activation loop that are conserved among each PIP kinase subfamily are shaded in green (type I), red (type II), and yellow (type III). In each case, identical amino acids are shaded in dark and similar amino acids are shaded in light colors. Accession numbers for sequences shown are: human Iα, Iβ, and mouse Iγ (Z29090, U79143, and BAA25664), D. melanogaster skittles (AAC25576), S. cerevisiae and S. pombe Mss4p (S61571, CAB10125), human IIα, IIβ, and mouse IIγ (NP_005019, NP_003550, and AAC40202), D. melanogaster type II (AI296694), S. cerevisiae, S. pombe, and C. elegans Fab1p (P34756, CAA19005, and CAA19436). Molecular Cell 2000 5, 1-11DOI: (10.1016/S1097-2765(00)80398-6)

Figure 2 PIP Kinase Reactions and HPLC Analysis of the Deacylated Glycerophosphorylinositol Products (A) Lipid kinase activities of Iβ (I), IIβ (II), and the IIβ–Iβloop (II*) and Iβ–IIβloop (I*) chimeras toward the synthetic phosphoinositide substrates indicated on top of each panel. PIP kinases were expressed in E. coli BL21(λDE3) as 6His fusion proteins, purified on Ni2+ beads, and tested for kinase activity against synthetic substrates. Phosphorylated phospholipids were separated by thin-layer chromatography. An autoradiogram of the kinase assay is shown. The positions of PIP2 and lysoPIP2 are indicated by arrows. (B) The deacylated products (broken lines) of PIP kinase reactions were analyzed using HPLC as described in the Experimental Procedures with [3H]GPIns, [3H]GPIns(3)P, [3H]GPIns(4)P, [3H]GPIns(3,5)P2, and [3H]GPIns(4,5)P as internal standards (solid lines). The peaks denoted with a star (*) result from contaminants present in the [32P]γATP. Molecular Cell 2000 5, 1-11DOI: (10.1016/S1097-2765(00)80398-6)

Figure 3 The IIβ–Iβloop Chimera Confers Type I Function In Vivo and Rescues the Lethality of the mss4–2ts Mutant Growth of mss4–2ts cells transformed with vector alone or vector containing MSS4, Iα, the IIβ–Iβloop chimera, or IIβ. Transformants were grown on YPG plates at 24°C (lower plate) or 38°C (upper plate) for 3–4 days. The slower growth of yeast cells containing MSS4 is likely due to the lower expression of MSS4 from its endogenous promoter compared to that of mammalian kinases from the Gal1 promoter. Molecular Cell 2000 5, 1-11DOI: (10.1016/S1097-2765(00)80398-6)

Figure 4 The IIβ–Iβloop Chimera Substitutes for Mss4p in the Regulation of Actin Cytoskeleton Remodeling and Cell Wall Integrity in Yeast Assay for suppression of the mss4–2ts-associated actin polarization, cell size, and cell integrity defects by either vector alone, or vector containing the IIβ–Iβloop chimera, MSS4, Iα, or IIβ. Three independent transformants each were grown in selective SG medium to early logarithmic phase at 24°C and then shifted to 38°C for 8 hr. Cells were fixed in formaldehyde solution, and the actin cytoskeleton was visualized with Texas Red–conjugated phalloidin. Cells were visualized by Nomarski optics. Shown are representative samples at the same magnification. Molecular Cell 2000 5, 1-11DOI: (10.1016/S1097-2765(00)80398-6)

Figure 5 The Activation Loop of Iβ Determines Targeting Specificity to the Plasma Membrane and In Vivo Function Human osteosarcoma MG-63 cells were transiently transfected with FLAG-Iβ (A–D), FLAG-Iβ–IIβloop (E–H), FLAG-IIβ–Iβloop (I–L), or FLAG-IIβ (M–P). Cells were fixed after 16 hr with paraformaldehyde, and the kinases were visualized with mouse monoclonal anti-FLAG antibody (A, C, E, G, I, K, M, and O), whereas F-actin was counterstained with Texas Red–conjugated phalloidin (B, D, F, H, J, L, N, and P). Images in the left-hand two panels are projections of multiple confocal sections, whereas images on the right-hand two panels represent a single confocal section to visualize the intracellular localization of the FLAG-tagged kinase. Sites of actin assembly are indicated by arrows. Molecular Cell 2000 5, 1-11DOI: (10.1016/S1097-2765(00)80398-6)

Figure 6 Mutation of the KK Motif Abolishes Plasma Membrane Association and Actin Reorganization (A) Segments of the activation loops of type I PIP kinases were aligned with PIP2-binding motifs in PH domains. Amino acid residues that are identical and similar in the majority of the proteins are shaded in black and grey, respectively. The alignment does not imply similar three-dimensional structure. (B) Human osteosarcoma MG-63 cells were transiently transfected with FLAG-Iβ(NN) (a–d), FLAG-Iβ(RR) (e–h), or FLAG-Iβ(kin−) (i–l). Cells were fixed after 16 hr with paraformaldehyde, and the kinases were visualized with mouse monoclonal anti-FLAG antibody antibody (a, c, e, g, i, and k), whereas F-actin was counterstained with Texas Red–conjugated phalloidin (b, d, f, h, j, and l). Images in the left-hand two panels are projections of multiple confocal sections, whereas images on the right-hand two panels represent a single confocal section to visualize the intracellular localization of the FLAG-tagged kinase. Sites of actin assembly are indicated by arrows. Molecular Cell 2000 5, 1-11DOI: (10.1016/S1097-2765(00)80398-6)