Stereo-specific substrate recognition by phosphatidylinositol phosphate kinases is swapped by changing a single amino acid residue

Jeannette Kunz, Allison Fuelling, Lottie Kolbe, Richard A. Anderson

Research output: Contribution to journalArticle

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Abstract

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 IIβ and the corresponding mutation E362A in Iβ, 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 Iβ(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 Iβ 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.

Original languageEnglish
Pages (from-to)5611-5619
Number of pages9
JournalJournal of Biological Chemistry
Volume277
Issue number7
DOIs
Publication statusPublished - Feb 15 2002
Externally publishedYes

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Phosphatidylinositol Phosphates
Phosphotransferases
Amino Acids
Substrates
1-Phosphatidylinositol 4-Kinase
Chemical activation
Second Messenger Systems
Cell membranes
Substrate Specificity
Phosphatidylinositols
Cell Membrane
Lipids
Mutagenesis
Amino Acid Substitution
Site-Directed Mutagenesis
Hydroxyl Radical
Substitution reactions
Cells
Mutation
Enzymes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Stereo-specific substrate recognition by phosphatidylinositol phosphate kinases is swapped by changing a single amino acid residue. / Kunz, Jeannette; Fuelling, Allison; Kolbe, Lottie; Anderson, Richard A.

In: Journal of Biological Chemistry, Vol. 277, No. 7, 15.02.2002, p. 5611-5619.

Research output: Contribution to journalArticle

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