Mek kinases: Dimers dim MAPK signaling

Mek1-Mek2 heterodimer formation is essential for Erk-mediated attenuation of the Raf-Mek-Erk signaling cascade.

The Raf–Mek–Erk signaling cascade is activated in response to growth factors and cell adhesion. The small GTPase Ras stimulates Raf to phosphorylate Mek1 and Mek2, which in turn phosphorylate and activate Erk1 and Erk2. This cascade affords rapid and tightly regulated signal amplification, but also contains a negative-feedback loop to attenuate signaling via Erk-mediated phosphorylation of Mek1 at Thr 292 (Mek2 lacks the analogous Thr residue). A report in Nature Structural and Molecular Biology by Manuela Baccarini and colleagues further elucidates this feedback mechanism by showing that Mek1 and Mek2 form heterodimers, and that dimer formation is necessary for the negative-feedback loop.

Raf–Mek–Erk signaling is involved in cell migration, and emerging evidence points to a distinct role for the two Mek isoforms. To investigate the importance of Mek1 in migration, primary fibroblasts were isolated from Mek1^-/- embryos. Surprisingly, these fibroblasts displayed enhanced Erk phosphorylation and migrated more rapidly towards mitogens than wild-type cells. Loss of Mek1 also caused prolonged and increased Raf-mediated phosphorylation of Mek2, and the authors showed that Mek1 normally restricts Mek2 phosphorylation. Mek1^-/- embryos displayed higher levels of Erk phosphorylation than heterozygotes, and targeted ablation of Mek1 in neuronal and epidermal tissue increased Mek and Erk activation. These data suggest that Mek1 restrains Mek signaling and support distinct and non-redundant roles for Mek1 and Mek2 in several cell and tissue types.

Mek1 and Mek2 are known to form homodimers. However, immunoprecipitation assays revealed that Mek1 and Mek2 also form a stable heterodimer complex. Point mutations in Mek1 that disrupted dimer formation without affecting Mek1 phosphorylation or kinase activity caused prolonged growth-factor-induced Erk phosphorylation. But how, then, does the Mek dimer affect Erk activation? Expression of a Mek1^T292A mutant that was resistant to the inhibitory Erk-mediated phosphorylation phenocopied Mek1 deficiency, whereas expression of a phosphomimetic Mek1^T292D mutant decreased Mek phosphorylation and led to faster Erk inactivation. Thus, heterodimer formation is essential for Erk-mediated phosphorylation of Thr 292 of Mek1, which attenuates Mek signaling.

Mek heterodimer formation underscores a key regulatory role for Mek1 in Raf–Mek–Erk signaling, and explains how Mek1 phosphorylation at Thr 292 can downregulate the activity of both Mek isoforms. These data also reinforce the importance of dimerization in mitogen-activated protein kinases. It will be interesting to examine whether dimers of other pathway members — such as the Raf kinases — undergo a similar means of negative feedback.

Emily J. Chenette
Signaling Gateway

Original Reference:
Catalanotti, F., Reyes, G. et al.
A Mek1–Mek2 heterodimer determines the strength and duration of the Erk signal
Nature Structural & Molecular Biology 16, 294-303 (2009)
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Cell cycle regulation: Akt Skps through the cell cycle

Akt-mediated phosphorylation of Skp2 promotes E3 ubiquitin ligase complex formation and stimulates p27 ubiquitylation and degradation.

Cell cycle progression is regulated in part by the ubiquitylation and degradation of the cyclin-dependent-kinase inhibitor p27. Skp2 (S-phase kinase-associated protein 2) is a substrate-recognizing subunit of the SCF (Skp1/Cul-1/F-box) E3 ubiquitin ligase that ubiquitylates p27. Indeed, overexpression of Skp2 induces cell cycle entry, and cytoplasmic mislocalization of Skp2 has been detected in human cancers. The regulation of Skp2 localization and its incorporation into the SCF complex remains an area of active investigation. Two complementary studies in Nature Cell Biology now show that Akt-mediated phosphorylation of Skp2 promotes its accumulation in the cytoplasm and increases SCF complex formation, resulting in p27 ubiquitylation and degradation.

Skp2 is known to be phosphorylated at the G1/S transition of the cell cycle, but the importance of this modification was not clear. Lin et al. found that Akt phosphorylated Skp2 at Ser 72 in vivo, which increased SCF complex formation and promoted the ubiquitylation and degradation of p27. Expression of wild-type or phosphomimetic, but not phosphorylation-deficient Skp2, also enhanced the proliferation and tumorigenic potential of a prostate cancer cell line.

Gao et al. showed that inhibition of PI(3)K-Akt signaling decreased Skp2 expression and confirmed that Skp2 was a direct substrate of Akt1, but not Akt2. Interestingly, Akt-mediated phosphorylation of Skp2 at Ser 72 stimulated subsequent phosphorylation at Ser 75 by casein kinase I. It was known that the E3 ubiquitin ligase complex APC-Cdh1 mediates Skp2 destruction, and phosphorylation at both these residues inhibited its interaction with Cdh1. Phosphomimetic Skp2 mutants were also resistant to APC-Cdh1-induced destruction.

Both groups reported that phosphorylated Skp2 is retained in the cytoplasm. Gao et al. found that phosphorylation of Skp2 impaired its binding to nuclear importin receptors, whereas Lin et al. detected an interaction between phosphorylated Skp2 and the cytoplasmic scaffolding protein 14-3-3β. Importantly, Akt phosphorylation and low expression of the PI(3)K antagonist PTEN (phosphatase and tensin homolog) correlated with the cytoplasmic localization of Skp2 in human prostate and colon cancer samples. In addition, pharmacologic inhibition of PI(3)K-Akt signaling blocked Skp2-mediated ubiquitination of p27, pointing to a potential avenue for therapeutic intervention in human cancer.

These results establish Skp2 as an important link between PI3K-Akt signaling and cell cycle progression. Intriguingly, cytoplasmic mislocalization of Skp2 was important for cell migration — and hence metastasis — that was independent of either the SCF complex or p27 degradation. It will be interesting to determine how Skp2 contributes to cell motility.

Emily J. Chenette
Signaling Gateway

Original References:
Lin, H.-K. et al.
Phosphorylation-dependent regulation of cytosolic localization and oncogenic function of Skp2 by Akt/PKB
Nature Cell Biology advance online publication, 8 March 2009 (DOI 10.1038/ncb1849)
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Gao, D., Inuzuka, H., Tseng, A., Chin, R. Y., Toker, A. & Wei, W.
Phosphorylation by Akt1 promotes cytoplasmic localization of Skp2 and impairs APC-Cdh1-mediated Skp2 destruction
Nature Cell Biology advance online publication, 8 March 2009 (DOI 10.1038/ncb1847)
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Calcium signaling: InsP₃ hosts a receptor get-together

The sensitivity of InsP₃ receptors is modulated by InsP₃-mediated receptor clustering.

Intracellular Ca²⁺ concentration is regulated in part by the activity of inositol-1,4,5-trisphosphate (InsP₃) receptors (InsP₃Rs). InsP₃R tetramers function as calcium channels that release Ca²⁺ from the endoplasmic reticulum in response to InsP₃ and Ca²⁺. Low concentrations of InsP₃ can stimulate a 'blip' of Ca²⁺ release from a single InsP₃R channel; the Ca²⁺ released then stimulates additional InsP₃Rs, generating a 'puff' of Ca²⁺. This puff of Ca²⁺ feeds forward to stimulate subsequent waves of Ca²⁺ release as InsP₃ and Ca²⁺ concentrations increase. However, the biochemical mechanism that underlies receptor clustering and the escalating response to InsP₃ and Ca²⁺ is not fully understood. Colin Taylor and colleagues now report in Nature that InsP₃ promotes receptor clustering, which tunes receptor sensitivity to InsP₃ and Ca²⁺ to regulate channel activity.

InsP₃R activity was studied in patches of outer nuclear envelope membranes. At resting Ca²⁺ concentrations, InsP₃ treatment caused InsP₃Rs to assemble reversibly into clusters of about four InsP₃Rs. However, these clusters exhibited decreased channel activity and were open for half as long as single InsP₃Rs. The reduced activity of clustered InsP₃Rs was not due to Ca²⁺ passing through active neighbors, suggesting that InsP₃ brings InsP₃Rs into physical contact, which attenuates their responses to InsP₃. Clustering decreased the distance between InsP₃Rs from an average of 1 μm to 20 nm — whereas single InsP₃Rs are insulated from neighboring channel activity, clustered InsP₃Rs are immediately exposed to the Ca²⁺ that is released by their neighbors. It remains to be determined whether the biochemical properties of InsP₃ binding are different for single channels compared with clusters of channels.

What, then, is the purpose of receptor clustering? Clustering ensures that Ca²⁺ released by one InsP₃R can regulate its neighbors. In addition, in a cluster, Ca²⁺ both reversed the diminished InsP₃ sensitivity and promoted coupled gating. If one channel opened, the other channel also opened, and these simultaneously open states were prolonged. Thus, clustering ensures that InsP₃Rs are more likely to be exposed to Ca²⁺ and exaggerates their responses to Ca²⁺.

These data provide insight into how InsP₃R activity is coordinated by InsP₃ and Ca²⁺. At resting Ca²⁺ concentrations, InsP₃ drives InsP₃R clustering, which restrains the release of Ca²⁺ but primes InsP₃R for Ca²⁺-mediated activity. Blips of Ca²⁺ release are then amplified into subsequent puffs and waves owing to the unique biochemical properties that clustering imparts on InsP₃Rs. It will be interesting to resolve the precise structural events that decrease the response to InsP₃ but increase Ca²⁺ sensitivity when InsP₃Rs are assembled in clusters.

Emily J. Chenette
Signaling Gateway

Original Reference:
Taufiq-Ur-Rahman, Skupin, A., Falcke, M. & Taylor, C. W.
Clustering of InsP₃ receptors by InsP₃ retunes their regulation by InsP₃ and Ca²⁺
Nature advance online publication, 25 February 2009 (DOI 10.1038/nature07763)
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Cancer stem cells: Killing Hedgehog to treat CML

Inhibition of the Hedgehog signaling pathway blocks chronic myelogenous leukemia progression by depleting cancer stem cells.

Most cases of chronic myeloid leukemia (CML) are caused by a chromosomal translocation that creates a fusion protein between breakpoint cluster region (BCR) and the tyrosine kinase ABL1. The tyrosine kinase inhibitor imatinib blocks CML progression; however, patients who harbor a BCR-ABL1^T315I point mutation are resistant to this drug and other tyrosine kinase inhibitors. A report in Nature now identifies a potential new therapeutic strategy for this disease. Tannishtha Reya and colleagues have shown that inhibition of the hedgehog (Hh) signaling pathway reduces the number of normal and malignant hematopoietic stem cells (HSCs) and blocks CML propagation.

To evaluate the role of Hh signaling in CML, Reya and colleagues developed mice that lacked expression of smoothened (SMO), the transmembrane protein that transmits Hh signals, in hematopoietic cells. The frequency of HSCs and differentiated cells was unaffected by SMO loss, but HSC renewal was severely impaired. Furthermore, the loss of SMO decreased cancer incidence and increased latency in a mouse model of CML, whereas transgenic expression of an activated form of SMO increased the number of CML cancer stem cells and accelerated cancer progression. Thus, Hh signaling positively regulates normal and malignant stem cell propagation.

Cyclopamine, which stabilizes inactive SMO and inhibits Hh signaling, reduced the ability of primary human blast crisis CML cells and mouse CML stem cells to form colonies in vitro. In addition, irradiated mice transplanted with BCR-ABL1-expressing HSCs had a reduced CML stem cell population and prolonged survival when treated with cyclopamine. Importantly, cyclopamine also slowed the progression of imatinib-resistant CML in mice and reduced colony formation of imatinib-resistant human CML cell lines. Therefore, targeted inhibition of Hh signaling could be useful for overcoming imatinib resistance in CML.

This study provides evidence that Hh inhibition impedes CML progression by affecting CML stem cells. Hh signaling is active in several hematological malignancies, and it will be important to determine whether inhibition of Hh signaling is beneficial in these diseases and solid tumors. Intriguingly, the authors found that Smo^-/- CML stem cells expressed higher levels of the Notch pathway inhibitor NUMB, and ectopic expression of NUMB blocked their propagation. As the authors' previous studies linked high levels of NUMB to HSC differentiation, these data suggest that NUMB expression as a result of inhibiting Hh may contribute to the depletion of cancer stem cells by promoting differentiation. The mechanistic details that link SMO loss to NUMB expression await further study.

Emily J. Chenette
Signaling Gateway

Original Reference:
Zhao, C., Chen, A. et al.
Hedgehog signalling is essential for maintenance of cancer stem cells in myeloid leukaemia
Nature advance online publication, 25 January 2009 (DOI 10.1038/nature07737)
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