PKA-site Phosphorylation Of Importin 13 Regulate Its Subcellular Localization And Nuclear Transport Function

Nucleocytoplasmic transport of proteins,RNAs,and ribosomes is integral to eukaryotic cell function.Importin ? family members interact with and import and/or export cargo proteins and certain RNAs across the nuclear envelope.There have been 20 genes encoding importin ? family members identified in the human genome.Importin 13(IP013)is a key member of the importin ? superfamily which can transport cargoes both into and out of the nucleus to contribute to a variety of important cellular processes.Phosphorylation is most widely characterized post-translation modification which can regulate various biological processes.It can also increase or impair the interaction between cargos and some importin ? superfamily members,result in the impact of physiological processes.cAMP-dependent protein kinase(PKA)is an enzyme playing key role in a number of cellular processes which can be activated by cAMP.It plays an important in regulation of cell cycle and proliferation,metabolism,transmission of nerve impulses,cytoskeleton remodeling,muscle contraction,cell survival and other cell processes.IP013 was detected to undergo phosphorylation by mass spectrum,but the impact of this on function has not been investigated.Here we show for the first time that IP013 is phosphorylated by cAMP-dependent protein kinase(PKA)specifically at serine 193 and made the specific site-phosphorylation antibody.We found this modification and mutation to mimic phosphorylation can induce IP013 cytoplasmic distribution by confocal laser scanning microscopy.Results from fluorescence recovery after photobleaching and fluorescence loss in photobleaching approaches establish that negative charge at serine 193 through phosphorylation or point mutation both reduces IP013 nuclear import,and increases its nuclear export.Phosphorylation also appears to enhance cargo interaction on the part of IP013,with significant impact on localization,as shown for the Pax6 homeobox-containing transcription partner.Endogenous IP013 can also be phosphorylated upon epinephrine induction which lead to cytoplasmic distribution.To investigate the other effect of IP013 phosphorylation,we detected the other post-translational modification and interaction protein after phosphorylation by mass spectrum.We also found that ERK1/2 pathway may be the downstream signal to affect IP013.This is the first report that IP013 can be phosphorylated at Ser193 and this modification regulates IP013 subcellular localization and nucleocytoplasmic transport function which affect the interaction and function of its cargos with important implications for IPO13's role in development and other processes.