Receptor-dependent compartmentalization of PPIP5K1, a kinase with a cryptic polyphosphoinositide binding domain

Abstract

The inositol pyrophosphates are multifunctional signalling molecules. One of the families of enzymes that synthesize the inositol pyrophosphates are the Vip1/PPIP5Ks (PP-InsP5 kinases). The kinase domains in Vip1/PPIP5Ks have been mapped to their N-terminus. Each of these proteins also possess a phosphatase-like domain of unknown significance. In the present study, we show that this phosphatase-like domain is not catalytically active. Instead, by using SPR (surface plasmon resonance) to study protein binding to immobilized lipid vesicles, we show that this domain is specialized for binding PtdIns(3,4,5)P3 (PPIP5K1 Kd = 96 nM; PPIP5K2 Kd = 705 nM). Both PtdIns(3,4)P2 and PtdIns(4,5)P2 are significantly weaker ligands, and no significant binding of PtdIns(3,5)P2 was detected. We confirm the functional importance of this domain in inositol lipid binding by site-directed mutagenesis. We present evidence that the PtdIns(3,4,5)P3-binding domain is an unusual hybrid, in which a partial PH (pleckstrin homology) consensus sequence is spliced into the phosphatase-like domain. Agonist-dependent activation of the PtdIns 3-kinase pathway in NIH 3T3 cells drives translocation of PPIP5K1 from the cytosol to the plasma membrane. We have therefore demonstrated receptor-regulated compartmentalization of inositol pyrophosphate synthesis in mammalian cells.

The Vip1 Inositol Polyphosphate Kinase Family Regulates Polarized Growth and Modulates the Microtubule Cytoskeleton in Fungi

“…Generation of inositol pyrophosphates has been shown for the budding yeast and human Vip1 family members [43]–[45], [48]. In this work we have extended the analysis to two further fungal Vip1-like proteins: the S. pombe Asp1 and the A. nidulans VlpA.…”

“…A recent publication has shown that the phosphatase-like domains of the human Vip1 members are catalytically inactive. Instead the authors show that this domain plays a role in inositol lipid binding [48]. On the other hand, a comparison of the amounts of inositol pyrophosphates generated by human and the S. cerevisiae full-length Vip1 proteins versus N-terminal kinase-domain-only-variants, showed that the latter variants exhibited more specific activity [43], [45].…”

The enzymes of human diphosphoinositol polyphosphate metabolism

“…A proteomics prediction that PPIP5K1 can be found in the nucleus [100] is contradicted by the finding that it is excluded from the nucleus of NIH 3T3 and HEK293 cells [97,99]. Phosphorylations of PPIP5K1 at Ser475, Tyr730, Ser944 and Ser1152 have been reported [101–103].…”

Inositol Pyrophosphates: Energetic, Omnipresent and Versatile Signalling Molecules

“…75 Both the PPIP5K isoforms possess a lipid inositide binding domain distinct from their kinase domains, and agonist-stimulated production of PI(3,4,5)P 3 can lead to translocation of PPIP5K1 from the cytoplasm to the plasma membrane. 41,76 As suggested by the ubiquitous expression and localisation of the kinases responsible for their synthesis, PP-IPs have been shown to participate in a myriad functions in many different tissues and subcellular compartments. This section describes several functions of these small molecules in different locations within a cell (Fig.…”