Stereo-specific Substrate Recognition by Phosphatidylinositol Phosphate Kinases Is Swapped by Changing a Single Amino Acid Residue*

By J. Kunz, Allison Fuelling, Lottie Kolbe, R. Anderson. 2002

84 citations recorded by the source corpus.

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.

Subfields: Cell Biology, Molecular Biology

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Citations in the corpus

In B cells, phosphatidylinositol 5-phosphate 4-kinase–α synthesizes PI(4,5)P2 to impact mTORC2 and Akt signaling (2016, methodology): “(11) that a single mutation (A381E) in the activation loop of human PI5P4Kβ changes its specificity from a PI5P4K”
The lipid kinase PI5P4Kβ is an intracellular GTP sensor for metabolism and tumorigenesis (2016, unknown): “Their ATP-dependent kinase activities were also confirmed, as previously reported (Demian et al., 2009; Kunz et al., 2002).”
Phosphatidylinositol 5-phosphate 4-kinase (PIP4K) regulates TOR signaling and cell growth during Drosophila development (2013, background): “1A, arrow), which is reported to define the stereospecific activity of a PIPK enzyme (12), revealed that the equivalent residue in CG17471 is an alanine rather than a glutamate.”