Identification of a functional nuclear translocation sequence in hPPIP5K2

Abstract

BackgroundCells contain several inositol pyrophosphates (PP-InsPs; also known as diphosphoinositol polyphosphates), which play pivotal roles in cellular and organismic homeostasis. It has been proposed that determining mechanisms of compartmentation of the synthesis of a particular PP-InsP is key to understanding how each of them may exert a specific function. Human PPIP5K2 (hPPIP5K2), one of the key enzymes that synthesizes PP-InsPs, contains a putative consensus sequence for a nuclear localization signal (NLS). However, such in silico analysis has limited predictive power, and may be complicated by phosphorylation events that can dynamically modulate NLS function. We investigated if this candidate NLS is functional and regulated, using the techniques of cell biology, mutagenesis and mass spectrometry.ResultsMultiple sequence alignments revealed that the metazoan PPIP5K2 family contains a candidate NLS within a strikingly well-conserved 63 amino-acid domain. By analyzing the distribution of hPPIP5K2-GFP in HEK293T cells with the techniques of confocal microscopy and imaging flow cytometry, we found that a distinct pool of hPPIP5K2 is present in the nucleus. Imaging flow cytometry yielded particular insight into the characteristics of the nuclear hPPIP5K2 sub-pool, through a high-throughput, statistically-robust analysis of many hundreds of cells. Mutagenic disruption of the candidate NLS in hPPIP5K2 reduced its degree of nuclear localization. Proximal to the NLS is a Ser residue (S1006) that mass spectrometry data indicate is phosphorylated inside cells. The degree of nuclear localization of hPPIP5K2 was increased when S1006 was rendered non-phosphorylatable by its mutation to Ala. Conversely, a S1006D phosphomimetic mutant of hPPIP5K2 exhibited a lower degree of nuclear localization.ConclusionsThe current study describes for the first time the functional significance of an NLS in the conserved PPIP5K2 family. We have further demonstrated that there is phosphorylation of a Ser residue that is proximal to the NLS of hPPIP5K2. These conclusions draw attention to nuclear compartmentation of PPIP5K2 as being a physiologically relevant and covalently-regulated event. Our study also increases general insight into the consensus sequences of other NLSs, the functions of which might be similarly regulated.Electronic supplementary materialThe online version of this article (doi:10.1186/s12860-015-0063-7) contains supplementary material, which is available to authorized users.

Inositol Trisphosphate Kinase and Diphosphoinositol Pentakisphosphate Kinase Enzymes Constitute the Inositol Pyrophosphate Synthesis Pathway in Plants

“…This suggests that the AtVIP PD might contain a nuclear export signal (NES), since the AtVIP-KD constructs were able to access the nucleus in the absence of the PD. We note that a nuclear localization sequence (NLS) has been identified near the C-terminus of the human PPIP5K2, and mutation of this NLS increases the portion of PPIP5K2 that accumulates in the cytoplasm of mammalian cells (Yong et al, 2015).…”

Intimate connections: Inositol pyrophosphates at the interface of metabolic regulation and cell signaling

“…Reciprocal regulation of the competing catalytic activities of PPIP5Ks is a particularly sensitive mechanism (Newsholme, 1971), by which quite small fluctuations in [Pi] can exert proportionately larger effects upon 1,5-InsP 8 levels (Gu et al, 2017). Since PPIP5Ks are predominantly located in the cytoplasm (Yong et al, 2015), that is where such Pi-sensing would have to occur. Unfortunately, cytoplasmic Pi homeostasis is not a well-studied topic, due to considerable technical hurdles.…”

The inositol pyrophosphate pathway in health and diseases

“…PPIP5K2 contains a penta-arginine (CPA) nuclear-localization sequence in the IDR domain (Fig. 4) (Yong et al, 2015). Overexpressed green fluorescent protein (GFP)-tagged CPA domain is nuclear, and phosphorylation of a serine residue adjacent to the domain regulates its nuclear localization (Yong et al, 2015).…”

“…4) (Yong et al, 2015). Overexpressed green fluorescent protein (GFP)-tagged CPA domain is nuclear, and phosphorylation of a serine residue adjacent to the domain regulates its nuclear localization (Yong et al, 2015). The impacts of phosphorylation of DIPP3 α at multiple C-terminal residues are unknown (Gauci et al, 2009).…”