Stereo-specific substrate recognition by phosphatidylinositol phosphate kinases is swapped by changing a single amino acid residue.
Type I and type II phosphatidylinositol phosphate (PIP) kinases generate the lipid second messenger phosphatidylinositol (PtdIns) 4,5-bisphosphate and thus play fundamental roles in the regulation of many cellular processes. Although the two kinase families are highly homologous, they phosphorylate distinct substrates and are functionally non-redundant. Type I PIP kinases phosphorylate PtdIns 4-phosphate at the D-5 hydroxyl group and are consequently PtdIns 4-phosphate 5-kinases. By contrast, type II PIP kinases are PtdIns 5-phosphate 4-kinases that phosphorylate PtdIns 5-phosphate at the D-4 position. Type I PIP kinases, in addition, also phosphorylate other phosphoinositides in vitro and in vivo and thus have the potential to generate multiple lipid second messengers. To understand how these enzymes differentiate between stereoisomeric substrates, we used a site-directed mutagenesis approach. We show that a single amino acid substitution in the activation loop, A381E in IIbeta and the corresponding mutation E362A in Ibeta, is sufficient to swap substrate specificity between these PIP kinases. In addition to its role in substrate specificity, the type I activation loop is also key in subcellular targeting. The Ibeta(E362A) mutant and other mutants with reduced PtdIns 4-phosphate binding affinity were largely cytosolic when expressed in mammalian cells in contrast to wild-type Ibeta which targets to the plasma membrane. These results clearly establish the role of the activation loop in determining both signaling specificity and plasma membrane targeting of type I PIP kinases.
Kunz
Jeannette
J
Department of Pharmacology, University of Wisconsin Medical School, Madison, Wisconsin 53706, USA.
Fuelling
Allison
A
Kolbe
Lottie
L
Anderson
Richard A
RA
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D002462:Cell Membrane / Q000201:enzymology; D004926:Escherichia coli / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007700:Kinetics; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000235:genetics*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D000596:Amino Acids; D017853:Phosphotransferases (Alcohol Group Acceptor)