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Nucleocytoplasmic shuttling of the Golgi phosphatidylinositol 4-kinase Pik1 is regulated by 14-3-3 proteins and coordinates Golgi function with cell growth.

ABSTRACT: The yeast phosphatidylinositol 4-kinase Pik1p is essential for proliferation, and it controls Golgi homeostasis and transport of newly synthesized proteins from this compartment. At the Golgi, phosphatidylinositol 4-phosphate recruits multiple cytosolic effectors involved in formation of post-Golgi transport vesicles. A second pool of catalytically active Pik1p localizes to the nucleus. The physiological significance and regulation of this dual localization of the lipid kinase remains unknown. Here, we show that Pik1p binds to the redundant 14-3-3 proteins Bmh1p and Bmh2p. We provide evidence that nucleocytoplasmic shuttling of Pik1p involves phosphorylation and that 14-3-3 proteins bind Pik1p in the cytoplasm. Nutrient deprivation results in relocation of Pik1p from the Golgi to the nucleus and increases the amount of Pik1p-14-3-3 complex, a process reversed upon restored nutrient supply. These data suggest a role of Pik1p nucleocytoplasmic shuttling in coordination of biosynthetic transport from the Golgi with nutrient signaling.

SUBMITTER: Demmel L

PROVIDER: S-EPMC2262966 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Yeast phosphatidylinositol 4-kinase, Pik1, has essential roles at the Golgi and in the nucleus.

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The Rules and Functions of Nucleocytoplasmic Shuttling Proteins.

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Protein kinase Cα regulates the nucleocytoplasmic shuttling of KRIT1.

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Salicylic acid inducible nucleocytoplasmic shuttling of NPR1 fusion proteins in human cells.

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Project description:Karyopherin α 2 (KPNA2, importin α1), a transport factor shuttling between the nuclear and cytoplasmic compartments, is involved in the nuclear import of proteins and participates in cellular processes such as cell cycle regulation, apoptosis, and transcriptional regulation. However, it is still unclear which signaling regulates the nucleocytoplasmic distribution of KPNA2 in response to cellular stress. In this study, we report that oxidative stress increases nuclear retention of KPNA2 through alpha serine/threonine-protein kinase (AKT1)-mediated reduction of serine 62 (S62) phosphorylation. We first found that AKT1 activation was required for H2O2-induced nuclear accumulation of KPNA2. Immunoprecipitation and quantitative proteomic analysis revealed that the phosphorylation of KPNA2 at S62 was decreased under H2O2-induced oxidative stress. We showed that cyclin-dependent kinase 1 (CDK1), a kinase responsible for KPNA2 S62 phosphorylation, contributes to the localization of KPNA2 in the cytoplasm. AKT1 knockdown increased KPNA2 S62 phosphorylation and inhibited CDK1 activation. Furthermore, H2O2-induced AKT1 activation promoted nuclear KPNA2 interaction with nucleophosmin 1 (NPM1), resulting in attenuation of NPM1-mediated cyclin D1 gene transcription. Thus, we infer that the AKT1-CDK1 axis regulates the nucleocytoplasmic shuttling and function of KPNA2 through spatiotemporal regulation of KPNA2 S62 phosphorylation under oxidative stress conditions.

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PIK1, an essential phosphatidylinositol 4-kinase associated with the yeast nucleus.

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Identification and partial characterization of new cell density-dependent nucleocytoplasmic shuttling proteins and open chromatin.

Project description:The contact inhibition of proliferation (CIP) denotes the cell density-dependent inhibition of growth, and the loss of CIP represents a hallmark of cancer. However, the mechanism by which CIP regulates gene expression remains poorly understood. Chromatin is a highly complex structure consisting of DNA, histones, and trans-acting factors (TAFs). The binding of TAF proteins to specific chromosomal loci regulates gene expression. Therefore, profiling chromatin is crucial for gaining insight into the gene expression mechanism of CIP. In this study, using modified proteomics of TAFs bound to DNA, we identified a protein that shuttles between the nucleus and cytosol in a cell density-dependent manner. We identified TIPARP, PTGES3, CBFB, and SMAD4 as cell density-dependent nucleocytoplasmic shuttling proteins. In low-density cells, these proteins predominantly reside in the nucleus; however, upon reaching high density, they relocate to the cytosol. Given their established roles in gene regulation, our findings propose their involvement as CIP-dependent TAFs. We also identified and characterized potential open chromatin regions sensitive to changes in cell density. These findings provide insights into the modulation of chromatin structure by CIP.

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