Each week we showcase a hot new cell signaling article from a Nature Publishing Group journal. Free full text access to the paper will be maintained for three months, after which the research highlight will be accessible via the Updates page.
Brief sleep deprivation attenuates the accumulation of cAMP in the hippocampus of mice, which blocks long-term potentiation and memory formation.
Sleep deprivation exacts a serious toll on memory consolidation and learning by impairing hippocampal function. Despite the pervasiveness of insufficient sleep, little is known about the molecular mechanisms that link it to alterations within the hippocampus. In Nature, Ted Abel and colleagues now report that sleep deprivation attenuates cAMP signaling in the hippocampus, which blocks long-term potentiation (LTP) and memory formation in mice.
Hippocampal LTP can be described as a lasting increase in the strength of synaptic signal transmission, and shares many features in common with memory consolidation. The maintenance of many forms of LTP depends on cAMP–protein kinase A (PKA) signaling, transcription and translation. Mice that had been briefly deprived of sleep exhibited impaired LTP, which was resolved following rest. LTP induced as a consequence of forskolin (FSK)-mediated adenylyl cyclase activation was also attenuated in sleep-deprived mice. Furthermore, sleep deprivation reduced cAMP levels, as well as phosphorylation of the cAMP pathway target CREB, in the hippocampus but not in other brain regions. Thus, insufficient sleep appears to reduce cAMP levels and therefore block LTP in the hippocampus.
Cyclic nucleotide phosphodiesterase (PDE) enzymes provide the only way of degrading cAMP. Intriguingly, insufficient sleep significantly increased the activity and levels of the PDE isoform PDE4A5 in hippocampal structures. Consistent with the hypothesis that PDE4A5 accumulation provides the molecular basis for lowered cAMP levels and LTP in sleep-deprived animals, the PDE4-selective inhibitor rolipram restored formation of LTP and rescued context-specific memory formation in sleep-deprived mice, but not in control mice, in vivo.
These data suggest that brief sleep deprivation induces the production of a specific PDE4 isoform in the hippocampus, which antagonizes cAMP accumulation and hence blocks LTP and memory formation. Whether long-term sleep deprivation affects these same signaling pathways will be interesting to explore. It will also be interesting to determine the extent to which hippocampal cAMP signaling is altered in people suffering from insufficient sleep, as phosphodiesterase inhibitors could have potential clinical use in restoring memory deficits caused by sleep deprivation.
Emily J. Chenette Signaling Gateway
Reference:
Vecsey, C. G. et al. Sleep deprivation impairs cAMP signalling in the hippocampus Nature461, 1122-1125 (2009) Full text | PDF | Subscribe to Nature
A phosphoproteomics approach has revealed that ERK-mediated phosphorylation of Nup50 inhibits the nuclear accumulation of importin-β.
High-throughput techniques aimed at elucidating all substrates of a protein kinase have been beset by difficulties. Phosphoproteomic assays may not uncover all substrates, and work done in vitro must be confirmed in vivo before a new substrate can be classified as such. In Nature Structural & Molecular Biology, Hidetaka Kosako et al. now apply a high-throughput approach to identify biologically relevant ERK substrates. This assay reveals that nucleoporin (Nup) proteins are direct ERK targets, elucidating a mechanism by which ERK modulates nucleo-cytoplasmic transport.
The phosphoproteome from cultured cells with selective activation of the B-Raf–MEK–ERK pathway was compared to that of ERK-inhibited cells by immobilized metal ion-affinity chromatography (IMAC) and two-dimensional difference gel electrophoresis (2D-DIGE). The resulting 'spots' were labeled with anti-P-X-pS-P and anti-pT-P antibodies that selectively recognize phosphorylated ERK consensus sequences. This combination of techniques should uncover direct ERK substrates. Indeed, many known ERK targets were identified; however, 24 proteins that are not known to be ERK substrates were also reported, including the nucleoporin Nup50.
Active ERK is known to inhibit nuclear import — so could Nup50 phosphorylation also be involved in this function? ERK was found to phosphorylate Nup50 in vitro and in vivo at Ser 221 and Ser 315, within the FG-repeat domain that binds importin-β. ERK also phosphorylated Nup153 and Nup214 within their importin-β-binding domains. Phosphorylation of these Nups did not affect nuclear pore complex assembly, but did attenuate their interactions with importin-β and transportin. Furthermore, ERK activation blocked the nuclear accumulation of importin-β and transportin, linking Nup phosphorylation to the regulation of nuclear import.
RNA interference (RNAi) experiments confirmed an essential role for Nup50 in ERK-mediated inhibition of importin-β nuclear import, whereas Nup153 was required for importin-β import in both ERK-stimulated and unstimulated cells. Rescuing Nup50 depletion with human NUP50, or a phosphomimetic NUP50 mutant, restored the ERK-mediated block. Thus, phosphorylation of Nup50 inhibits importin-β nuclear translocation following ERK pathway activation. It will be important to evaluate the extent to which phosphorylation of Nup proteins affects nuclear import as a whole, as importin-β shepherds many disparate proteins into the nucleus.
Emily J. Chenette Signaling Gateway
Reference:
Kosako, H. et al. Phosphoproteomics reveals new ERK MAP kinase targets and links ERK to nucleoporin-mediated nuclear transport Nature Structural & Molecular Biology16, 1026-1035 (2009) Full text | PDF | Subscribe to Nature Structural & Molecular Biology
Further reading:
Ahn, N. G. PORE-ing over ERK substrates Nature Structural & Molecular Biology16, 1004-1005 (2009) Full text | PDF | Subscribe to Nature Structural & Molecular Biology
Chemotherapy: Imatinib is Abl to preserve fertility
Pharmaceutical inhibition of a c-Abl–TAp63 signaling pathway protects female mice from chemotherapy-induced infertility.
Women undergoing chemotherapy treatment for cancer face a high risk of ovarian failure and infertility, as genotoxic stress can induce apoptosis in germ cells. In Nature Medicine, Stefania Gonfloni et al. now identify a pro-apoptotic c-Abl–TAp63 pathway that is induced by chemotherapy in oocytes. Pharmaceutical inhibition of c-Abl partially rescues fertility in cisplatin-treated mice.
The p53-related protein TAp63-α is required for the induction of irradiation-induced cell death in oocytes. The chemotherapeutic cisplatin, which induces DNA cross-links and apoptosis, caused a transient increase in TAp63 levels. Cisplatin is known to activate the c-Abl kinase, and the authors found that inhibition of c-Abl by the therapeutic tyrosine-kinase inhibitor imatinib prevented TAp63 accumulation and protected oocytes from cisplatin-induced cell death.
To investigate a potential functional interaction between c-Abl and TAp63, the p53-deficient Saos-2 cell line was stably transfected with TAp63 under the control of an inducible promoter. In these cells, cisplatin upregulated c-Abl expression, nuclear accumulation and activity, but only induced apoptosis when TAp63 was expressed. A constitutively active mutant of c-Abl phosphorylated TAp63 at three tyrosine residues; these residues were essential for the TAp63-mediated induction of pro-apoptotic genes.
Female mice treated with cisplatin delivered consistently smaller litter sizes and eventually became infertile. Indeed, cisplatin treatment caused a decrease in primordial and primary follicles in mouse ovaries. Remarkably, co-treatment with imatinib significantly increased the number of primordial and primary follicles, and partially rescued infertility.
These results highlight a novel c-Abl–TAp63 pathway that induces apoptosis in response to cisplatin, and suggest that inhibition of c-Abl during chemotherapy might help preserve fertility in women. A remaining question is whether inhibition of this pathway will affect tumor regression, especially given that p63 has a known role in cell death, and that activation of c-Abl has been shown to induce apoptosis in tumors. It will be important to determine if imatinib treatment during chemotherapy affects tumor regression, and if other DNA-damaging agents can also induce c-Abl–TAp63 activation.
Emily J. Chenette Signaling Gateway
References:
Gonfloni, S. et al. Inhibition of the c-Abl–TAp63 pathway protects mouse oocytes from chemotherapy-induced death Nature Medicine15, 1179-1185 (2009) Full text | PDF | Subscribe to Nature Medicine
Woodruff, T. K. Preserving fertility during cancer treatment Nature Medicine15, 1124-1125 (2009) Full text | PDF | Subscribe to Nature Medicine
Protein trafficking: PTEN hitches a ride on myosinV
An interaction between PTEN and myosinV regulates PTEN function and neuronal soma size.
The lipid phosphatase PTEN antagonizes PI(3)K (phosphoinositide 3-kinase) activity by dephosphorylating phosphatidylinositol (3,4,5)-trisphosphate (PIP3) to form PI(4,5)P2. PTEN is active at the plasma membrane, where the PI(3)K-generated pools of PIP3 reside, but the mechanisms governing PTEN membrane translocation are not well understood. In Nature Cell Biology, Britta Eickholt and colleagues now identify a previously unappreciated interaction between PTEN and the motor protein myosinV that regulates PTEN membrane association and neuronal soma size.
The myosinV family members myosinVa, myosinVb and myosinVc were identified as novel PTEN-interacting proteins in neurons. This association was dependent on phosphorylated serine and threonine residues in the C terminus of PTEN; inhibition of casein kinase 2 (CK2) or glycogen synthase kinase 3 (GSK3) attenuated the interaction. Fluorescence lifetime imaging microscopy (FLIM) detected co-localization of myosinVa and PTEN at the cell periphery, which was dependent on either CK2 or GSK3 activity.
Overexpression of the myosinVa globular domain (MVag), which competes with full-length myosinV proteins for binding to PTEN, increased soma size of murine hippocampal neurons in vivo — a phenotype reminiscent of hippocampal neurons in PTEN-null mice. This phenotype was reverted by treatment with the PI(3)K inhibitor LY294002 or the mTOR inhibitor rapamycin. Furthermore, the expression of a constitutively membrane-associated myristoylated version of PTEN overcame the phenotypic effects of MVag overexpression, suggesting that myosinV might regulate PI(3)K signaling by translocating PTEN to the plasma membrane.
Soma enlargement due to PTEN deficiency was previously shown to require PI(3)K–mTOR–S6 signaling. Eickholt and colleagues found that constitutively active GSK3β reduced S6 phosphorylation and soma size, whereas expression of either MVag or a phosphatase-dead myristoylated PTEN mutant increased soma size and S6 phosphorylation. A series of epistasis experiments revealed that GSK3β was upstream of myosinV and that PTEN catalytic activity was required for regulation of neuronal cell growth.
These data reveal that CK2 and GSK3β activity induces an interaction between PTEN and myosinV, which regulates PTEN membrane localization and restrains neuronal soma size. Intriguingly, GSK3β is known to be inhibited by PI(3)K signaling, which suggests that the PTEN–myosinV interaction might be regulated by a positive feedback loop. This possibility, and whether the PTEN–myosinV interaction is preserved in other cell types, await further studies.
Emily J. Chenette Signaling Gateway
References:
van Diepen, M. T., Parsons, M., Downes, C. P., Leslie, N. R., Hindges, R. & Eickholt, B. J. MyosinV controls PTEN function and neuronal cell size Nature Cell Biology11, 1191-1196 (2009) Full text | PDF | Subscribe to Nature Cell Biology
Zhou, J. & Parada, L. F. A motor driving PTEN Nature Cell Biology11, 1177-1179 (2009) Full text | PDF | Subscribe to Nature Cell Biology
β-catenin regulation: TNKS for nothing
A small-molecule inhibitor of tankyrase promotes the degradation of β-catenin by stabilizing axin protein levels.
In resting cells, β-catenin is phosphorylated and targeted for destruction by a complex containing adenomatous polyposis coli (APC), axin 1/2 and glycogen synthase kinase 3α/β (GSK3α/β). Wnt signaling causes the disassociation of this complex, permitting β-catenin to translocate to the nucleus and stimulate gene transcription. Dysregulation of APC, axin or GSK3α/β can cause cancer via unrestrained β-catenin activity, but small-molecule inhibitors of the Wnt pathway have been difficult to develop. In Nature, Feng Cong and colleagues now identify a novel Wnt pathway inhibitor that restrains β-catenin by stabilizing axin protein levels.
A high-throughput reporter assay for Wnt pathway modulators identified the compound XAV939, which increased axin protein levels, blocked nuclear β-catenin accumulation and inhibited Wnt-stimulated transcription in cultured cells. In zebrafish, XAV939 treatment antagonized Wnt-dependent regenerative processes. XAV939 binds to and inhibits the poly(ADP-ribose) polymerases tankyrase 1 (TNKS1) and TNKS2. Indeed, small interfering (siRNA)-mediated co-depletion of TNKS1 and TNKS2 increased axin protein levels, leading to phosphorylation and degradation of β-catenin, and inhibition of target gene transcription. The ankyrin-repeat domain of TNKS1 and the TNKS-binding domain (TBD) of axin interacted in vitro, and deletion of the TBD strongly upregulated axin levels. Remarkably, a role for TNKS in Wnt pathway signaling had not previously been appreciated.
TNKS enzymes catalyze PARsylation — the addition of poly-ADP-ribose — which often promotes the ubiquitination and proteasomal degradation of substrates. TNKS was found to PARsylate axin, which was blocked by XAV939. Treatment with a proteasome inhibitor stabilized axin levels, resulting in the accumulation of ubiquitylated and PARsylated axin; however, the pool of PARsylated axin disappeared when cells were also treated with XAV939. These results are consistent with the hypothesis that TNKS-mediated PARsylation of axin results in its subsequent ubiquitination and proteolytic degradation, and explain how TNKS inhibition might increase cellular axin levels.
Aberrant Wnt signaling is a hallmark of many colorectal cancers, but the current pharmaceutical armamentarium lacks a Wnt-specific inhibitor. XAV939 treatment had no effect on β-catenin-independent RKO colorectal cancer cells, but dramatically blocked the colony-forming ability of β-catenin-dependent DLD-1 colorectal cancer cells. siRNA against axin restored the colony-forming potential of DLD-1 cells. Thus, stabilization of axin by inhibition of TNKS1 and TNKS2 might be a novel therapeutic strategy in β-catenin-dependent colorectal cancers.
Emily J. Chenette Signaling Gateway
References:
Huang, S.-M. A. et al. Tankyrase inhibition stabilizes axin and antagonizes Wnt signaling Nature461, 614-620 (2009) Full text | PDF | Subscribe to Nature
Peterson, R. T. Drug discovery: Propping up a destructive regime Nature461, 599-600 (2009) Full text | PDF | Subscribe to Nature
