Type I phosphatidylinositol 4-phosphate 5-kinase controls neutrophil polarity and directional movement

Directional cell movement in response to external chemical gradients requires establishment of front–rear asymmetry, which distinguishes an up-gradient protrusive leading edge, where Rac-induced F-actin polymerization takes place, and a down-gradient retractile tail (uropod in leukocytes), where RhoA-mediated actomyosin contraction occurs. The signals that govern this spatial and functional asymmetry are not entirely understood. We show that the human type I phosphatidylinositol 4-phosphate 5-kinase isoform β (PIPKIβ) has a role in organizing signaling at the cell rear. We found that PIPKIβ polarized at the uropod of neutrophil-differentiated HL60 cells. PIPKIβ localization was independent of its lipid kinase activity, but required the 83 C-terminal amino acids, which are not homologous to other PIPKI isoforms. The PIPKIβ C terminus interacted with EBP50 (4.1-ezrin-radixin-moesin (ERM)-binding phosphoprotein 50), which enabled further interactions with ERM proteins and the Rho-GDP dissociation inhibitor (RhoGDI). Knockdown of PIPKIβ with siRNA inhibited cell polarization and impaired cell directionality during dHL60 chemotaxis, suggesting a role for PIPKIβ in these processes

An isoform-specific PDZ-binding motif targets type I PIP5 kinase beta to the uropod and controls polarization of neutrophil-like HL60 cells

Type I phosphatidylinositol 4-phosphate 5-kinase (PIP5KI)-β participates in establishing polarity during leukocyte chemotaxis. Its final 83 amino acids localize PIP5KIβ to the uropod of chemotaxing neutrophils and T cells, and interact with ezrin-radixin-moesin (ERM) proteins and EBP50 (4.1-ERM-binding phosphoprotein 50), a scaffold protein with 2 PDZ (PSD-95, disc large, ZO-1) domains. The structural motifs at the PIP5KIβ C terminus that confer signaling specificity are, nonetheless, unknown. We show that the last 4 residues of PIP5KIβ constitute an atypical PDZ-binding motif, which steers PIP5KIβ to the uropod by binding to both EBP50 PDZ domains. Molecular modeling and mutagenesis indicated that PDZ-binding motif is necessary for PIP5KIβ localization and for chemoattractant-induced neutrophil polarization. Polarity in cells that express PIP5KIβ mutants lacking the PDZ-binding motif was restored by overexpression of PIP5KIβ, but not of PIP5KIγ_i2, another isoform that localizes to the neutrophil uropod. Our results identify an isoform-specific PDZ-binding motif in PIP5KIβ, which confers specificity for PIP5KIβ signaling at the uropod during leukocyte chemotaxis.—Mañes, S., Fuentes, G., Peregil, R. M., Rojas, A. M., Lacalle, R. A. An isoform-specific PDZ-binding motif targets type I PIP5 kinase beta to the uropod and controls polarization of neutrophil-like HL60 cells.This work was supported, in part, by the Spanish Ministry of Science and Innovation (SAF2008-00649), the Carlos III Health Institute RIER network (RD08/0075/0007), the European Union FP6 (INNOCHEM, LSHB-CT-2005-518167), the ERAnet program (e-rare, PI07/1314), the Comunidad de Madrid (DIFHEMAT, S-SAL-0304-2006), and the Genoma España Foundation (MEICA).Peer reviewe

Type I phosphatidylinositol 4-phosphate 5-kinase homo- and heterodimerization determines its membrane localization and activity

Type I phosphatidylinositol 4-phosphate 5-kinases (PIP5KIs; α, β, and γ) are a family of isoenzymes that produce phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] using phosphatidylinositol 4-phosphate as substrate. Their structural homology with the class II lipid kinases [type II phosphatidylinositol 5-phosphate 4-kinase (PIP4KII)] suggests that PIP5KI dimerizes, although this has not been formally demonstrated. Neither the hypothetical structural dimerization determinants nor the functional consequences of dimerization have been studied. Here, we used Förster resonance energy transfer, coprecipitation, and ELISA to show that PIP5KIβ forms homo- and heterodimers with PIP5KIγ_i2 in vitro and in live human cells. Dimerization appears to be a general phenomenon for PIP5KI isoenzymes because PIP5KIβ/PIP5KIα heterodimers were also detected by mass spectrometry. Dimerization was independent of actin cytoskeleton remodeling and was also observed using purified proteins. Mutagenesis studies of PIP5KIβ located the dimerization motif at the N terminus, in a region homologous to that implicated in PIP4KII dimerization. PIP5KIβ mutants whose dimerization was impaired showed a severe decrease in PI(4,5)P2 production and plasma membrane delocalization, although their association to lipid monolayers was unaltered. Our results identify dimerization as an integral feature of PIP5K proteins and a central determinant of their enzyme activity.—Lacalle, R. A., de Karam, J. C., Martínez-Muñoz, L., Artetxe, I., Peregil, R. M., Sot, J., Rojas, A. M., Goñi, F. M., Mellado, M., Mañes, S. Type I phosphatidylinositol 4-phosphate 5-kinase homo- and heterodimerization determines its membrane localization and activity.J.C.d.K. is a Ph.D. fellow of the La Caixa Foundation International Fellowship Programme. This work was supported in part by the Spanish Ministry of Science and Innovation (SAF2011-24453) and the Comunidad de Madrid (IMMUNOTHERCAN; S2010/BMD-2326).Peer reviewe