Identification of a Nuclear Export Signal in the Catalytic Subunit of AMP-activated Protein Kinase

Kazgan, Nevzat; Williams, Tyisha; Forsberg, Lawrence J.; Brenman, Jay E.

doi:10.1091/mbc.e10-04-0347

Cited by 104 publications

(109 citation statements)

References 53 publications

(82 reference statements)

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“…AMPK is also modulated by CRM1-mediated export via its C-terminal NES (34). AMPK catalyzes the phosphorylation of several transcription factors (e.g., FoxO3 and PGC-1) (35,36).…”

Section: Resultsmentioning

confidence: 99%

AMPK Facilitates Nuclear Accumulation of Nrf2 by Phosphorylating at Serine 550

Joo

Kim

Lee

et al. 2016

Molecular and Cellular Biology

393

257

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b Nrf2 (nuclear factor erythroid 2-related factor 2) is an antioxidant transcription factor. AMP-activated protein kinase (AMPK) functions as a central regulator of cell survival in response to stressful stimuli. Nrf2 should be coordinated with the cell survival pathway controlled by AMPK, but so far the mechanistic connections remain undefined. This study investigated the role of AMPK in Nrf2 trafficking and its activity regulation. A subnetwork integrating neighbor molecules suggested direct interaction between AMPK and Nrf2. In cells, AMPK activation caused nuclear accumulation of Nrf2. In the in vitro kinase and peptide competition assays, AMPK phosphorylated Nrf2 at the Ser558 residue (Ser550 in mouse) located in the canonical nuclear export signal. Nrf2 with an S550A mutation failed to be accumulated in the nucleus after AMPK activation. Leptomycin B, a nuclear export inhibitor, did not enhance nuclear accumulation of wild-type Nrf2 (WT-Nrf2) activated by AMPK or a phospho-Ser550-mimetic Nrf2 mutant, corroborating the finding that AMPK facilitated nuclear accumulation of Nrf2, probably by inhibiting nuclear export. Activated glycogen synthase kinase 3␤ (GSK3␤) diminished the basal nuclear level of Myc-S550A-Nrf2. Taking the data collectively, AMPK phosphorylates Nrf2 at the Ser550 residue, which, in conjunction with AMPK-mediated GSK3␤ inhibition, promotes nuclear accumulation of Nrf2 for antioxidant response element (ARE)-driven gene transactivation. Redox homeostasis in the cell may be disturbed by a deficiency in energy production as well as by reactive oxygen species (ROS) generated from xenobiotic biotransformation or the process of fuel oxidation (1-3). Nrf2 (nuclear factor erythroid 2-related factor 2), a key antioxidant transcription factor, participates in maintaining redox homeostasis in the cell (4). Various physiological or pathological circumstances accompanying free radical generation activate Nrf2 as a consequence of an adaptive response to oxidative stress (5). However, the regulation of Nrf2 has been minimally studied in the context of cellular energy deficiency (e.g., starvation, hypoxia, and exercise).The response of Nrf2 to oxidative challenge stress rapidly occurs mainly through phosphorylation at Ser40; protein kinase C␦ (PKC␦) induces activation of phosphorylation of Nrf2 at the residue, and the phosphorylated form is then released from Keap-1 and is stabilized for antioxidant response element (ARE)-driven gene expression (6, 7). An active form of Nrf2 has been shown to localize in the nucleus and binds to the ARE(s) present in the promoter regions of target genes (8). In this process, phosphoinositide 3-kinase controls nuclear translocation of Nrf2 (9). In contrast, glycogen synthase kinase 3␤ (GSK3␤) catalyzes the inhibitory phosphorylation of Nrf2 for the tight activity control (10, 11). Other kinases, including mitogen-activated protein kinases and Fyn, may also affect Nrf2 activity (12, 13). Despite the identification of oxidative stress-associated Nrf2 kinases, the characterist...

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“…AMPK is also modulated by CRM1-mediated export via its C-terminal NES (34). AMPK catalyzes the phosphorylation of several transcription factors (e.g., FoxO3 and PGC-1) (35,36).…”

Section: Resultsmentioning

confidence: 99%

AMPK Facilitates Nuclear Accumulation of Nrf2 by Phosphorylating at Serine 550

Joo

Kim

Lee

et al. 2016

Molecular and Cellular Biology

393

257

View full text Add to dashboard Cite

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“…Moreover, a putative NES could also be present in the C-terminus of Ssp2. Recently, a functional NES in the C-terminal 22 amino acids of human AMPK a catalytic subunit has been described (Kazgan et al, 2010). The fission yeast Ssp2 C-terminal sequence matches the consensus sequence of highly enriched bulky hydrophobic amino acids (f5 Leu, Ile, Phe, Val and Met), with the following spacings (f-x-2/3-f-x-2/3-fx-f) in a helix (Kutay and Güttinger, 2005).…”

Section: Discussionmentioning

confidence: 99%

AMPK phosphorylation by Ssp1 is required for proper sexual differentiation in fission yeast

Valbuena¹,

Moreno²

2012

Journal of Cell Science

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SummaryThe AMP-activated protein kinase (AMPK) is a central regulator of cellular energy homeostasis, which, in response to a fall in intracellular ATP levels, activates energy-producing pathways and inhibits energy-consuming processes. Here, we report that fission yeast cells lacking AMPK activity are unable to advance entry into mitosis in response to nitrogen starvation and cannot undergo proper G1 arrest and cell differentiation. We also show that AMPK is important in the promotion of the nuclear localization and accumulation of the Ste11 transcription factor. As in animal cells, the fission yeast CaMKK ortholog (Ssp1) phosphorylates and activates the catalytic subunit of AMPK (Ssp2) in its activation loop (Thr189) when cells are starved for nitrogen or glucose. Interestingly, we found that the phosphorylation of Ssp2 on Thr189 is required for nuclear accumulation of AMPK. Our data demonstrate the existence of a signal transduction pathway activated by nutrient starvation that triggers Ssp2 phosphorylation and AMPK redistribution from the cytoplasm to the nucleus. This pathway is important to advance fission cells into mitosis and to establish a timely pre-Start G1 cell cycle arrest for mating.

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“…Both pmCherry-RanQ69L (human; 30309) and pTK21-Ran T24N (human; 37396) were purchased from Addgene (Addgene, Cambridge, MA). These constructs were separately contributed by the Jay Brenman (Kazgan et al, 2010) and Iain Cheeseman (Kiyomitsu and Cheeseman, 2012) laboratories. To ensure uniformity in the comparison of Ran mutants, the Ran T24N gene fragment was isolated from pTK21 and inserted into the pmCherry-C1 vector.…”

Section: Dna Constructsmentioning

confidence: 99%

A novel role for Rab23 in the trafficking of Kif17 to the primary cilium

Lim

Tang

2015

Journal of Cell Science

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The small GTPase Rab23 is an antagonist of sonic hedgehog (Shh) signaling during mouse development. Given that modulation of Shh signaling depends on the normal functioning of the primary cilium, and overexpression of Evi5L, a putative Rab23 GTPase-activating protein (GAP), leads to reduced ciliogenesis, Rab23 could have a role at the primary cilium. Here, we found that wild-type Rab23 and the constitutively active Rab23 Q68L mutant were enriched at the primary cilium. Therefore, we tested the role of Rab23 in the ciliary targeting of known cargoes and found that ciliary localization of the kinesin-2 motor protein Kif17 was disrupted in Rab23-depleted cells. Co-immunoprecipitation and affinity-binding studies revealed that Rab23 exists in a complex with Kif17 and importin β2 (the putative Kif17 ciliary import carrier), implying that Kif17 needs to bind to regulatory proteins like Rab23 for its ciliary transport. Although a ciliary-cytoplasmic gradient of nuclear Ran is necessary to regulate the ciliary transport of Kif17, Rab23 and Ran appear to have differing roles in regulating the ciliary entry of Kif17. Our findings have uncovered a hitherto unknown effector of Rab23 and demonstrate how Rab23 could mediate the transport of Kif17 to the primary cilium.

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Identification of a Nuclear Export Signal in the Catalytic Subunit of AMP-activated Protein Kinase

Cited by 104 publications

References 53 publications

AMPK Facilitates Nuclear Accumulation of Nrf2 by Phosphorylating at Serine 550

AMPK Facilitates Nuclear Accumulation of Nrf2 by Phosphorylating at Serine 550

AMPK phosphorylation by Ssp1 is required for proper sexual differentiation in fission yeast

A novel role for Rab23 in the trafficking of Kif17 to the primary cilium

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