Kinase cogs go forward and reverse in the Wnt ...

NEWS AND VIEWS 11. Liu, J., Valencia-Sanchez, M.A., Hannon, G.J. & Parker, R. Nat. Cell Biol. 7, 716–723 (2005). 12. Sen, G.L. & Blau, H.M. Nat. Cell Biol. 7, 633–636 (2005). 13. Sheth, U. & Parker, R. Science 300, 805–808 (2003). 14. Chen, C.Y. et al. Cell 107, 451–464 (2001). 15. Mukherjee, D. et al. EMBO J. 21, 165–174 (2002).

16. Lykke-Andersen, J. & Wagner, E.R. Genes Dev. 19, 351–361 (2005). 17. Jing, Q. et al. Cell 120, 623–634 (2005). 18. Kshirsagar, M. & Parker, R. Genetics 166, 729–739 (2004). 19. Badis, G., Saveanu, C., Fromont-Racine, M. & Jacquier, A. Mol. Cell 15, 5–15 (2004). 20. Coller, J. & Parker, R. Cell 122, 875–886 (2005).

Kinase cogs go forward and reverse in the Wnt signaling machine Trevor Dale An important link between Wnt binding at the cell surface and nuclear β-catenin–TCF–dependent transcription has been made with the identification of kinases that promote the association of the Wnt receptor and β-catenin turnover complexes. Surprisingly, the enzymes implicated had previously been suggested to inhibit rather than promote Wnt signaling.

Wnt

Wnt

LRP

CK1 P P P PP

P P P P PP

CK1 PPPSP Kinase (GSK-3)

P P P P PP

CK1 P P P PP

The author is at the Cardiff School of Biosciences, Biomedical Sciences Building, Museum Avenue, Cardiff, CF10 3US UK. e-mail: [email protected]

a

PP

Two recent papers have identified casein kinase 1 (CK1) as a key player in the stabilization of β-catenin by Wnt ligands1,2. In these studies, Wnt-induced phosphorylation of the Wnt coreceptor LRP6 by CK1 was shown to enhance the binding of Axin, a central component of the β-catenin turnover complex, to the receptor (Fig. 1a). This association is proposed to inhibit β-catenin turnover by as-yet-unidentified mechanisms, leading to raised levels of β-catenin and the activation of nuclear β-catenin– TCF–dependent transcription. The phosphorylation of LRP6 adds a new role for CK1 to a growing list of activities linking CK1 to Wnt signaling. Foremost amongst these is the kinase’s participation in β-catenin turnover. In this role, CK1 binds directly to Axin (or indirectly via Diversin) to promote β-catenin degradation by priming GSK-3– mediated phosphorylation of β-catenin, resulting in β-catenin ubiquitination and degradation3,4. By contrast, the new work on LRP phosphorylation suggests a role for the CK1 in suppressing β-catenin degradation, adding to several reports suggesting positive roles for CK1 in Wnt signaling through interactions with the intracellular protein Dishevelled and the nuclear transcription factor TCF (reviewed in ref. 5). Is there a way to reconcile these contradictory positive and negative roles? Much may

PP

© 2006 Nature Publishing Group http://www.nature.com/nsmb

7. Dunckley, T. & Parker, R. EMBO J. 18, 5411–5422 (1999). 8. She, M. et al. Nat. Struct. Mol. Biol. 11, 249–256 (2004). 9. Coller, J. & Parker, R. Annu. Rev. Biochem. 73, 861–890 (2004). 10. Cougot, N., Babajko, S. & Seraphin, B. J. Cell Biol. 165, 31–40 (2004).

Axin

b

Thr1479

Thr1493 Ser1490

A 1460-

-1505

cluster 1

B 1527C 1569D 1588E 1604-

-1533 -1574 -1596 -1613

cluster 2 Figure 1 Wnt-induced CK1 activity phosphorylates LRP6 and enhances binding by Axin. (a) Schematic showing phosphorylation and binding. The interaction between Axin and LRP is proposed to be crucial for the inhibition of Axin-templated β-catenin turnover. (b) Sequence of the LRP6 C terminus showing conserved residues in red. The five PPPSP motifs (A–E, ordered from the N to the C terminus) are boxed. Studies with phospho-specific antibodies show that CK1 phosphorylates residues Thr1479 and Thr1493, flanking PPPSP motif A. Phosphorylation of Thr1493 is primed by prephosphorylation of Ser1490. A similar priming relationship is predicted for analogous residues on the C-terminal sides of PPPSP motifs B–E, which are jointly referred to as cluster 2. CK1 is also thought to phosphorylate residues in cluster 1, on the N-terminal side of PPPSP motif A.

NATURE STRUCTURAL & MOLECULAR BIOLOGY VOLUME 13 NUMBER 1 JANURARY 2006

9

NEWS AND VIEWS i

iii

ii +Wnt

Inhibition of b-catenin turnover

+Axin binding Wnt

Wnt

Fz

Fz

LRP

PP P

CK1ε

CK1ε

PPPP PP

Dsh

Dsh PP

CK1γ PP PP PP

CK1γ

PPP

© 2006 Nature Publishing Group http://www.nature.com/nsmb

CK1γ

CK1α

Figure 2 Model of Wnt signaling involving functions of multiple CK1 isoforms. (i) CK1γ and the PPPSP kinase maintain a low basal level of LRP phosphorylation. (ii) Wnt ligand induces a complex with Frizzled and associated proteins. Kinases associated with Frizzled increase LRP phosphorylation. In the specific model illustrated, Dsh-associated CK1ε increases the proportion of LRP sites that are phosphorylated. (iii) The increased affinity of LRP for Axin promotes the association of the β-catenin turnover complex with LRP. This results in the further phosphorylation of LRP by Axin-associated kinases including CK1α, which is directly bound to Axin, and by CK1ε, which associates indirectly with Axin via Diversin (not shown)3. The tightly bound receptor–β-catenin turnover complex is unable to degrade β-catenin, leading to its accumulation and to the activation of β-catenin–TCF–dependent transcription.

come down to differences between CK1 subtypes and to the details of the phosphorylated residues in the LRP receptors that bind Axin (Fig. 1b). There are seven mammalian CK1 members: α, α2, β, δ, ε, γ1, γ2 and γ3 (ref. 6). Previous studies have shown that CK1α negatively regulates β-catenin as part of a complex centered on Axin7–9. Davidson et al.1 have now shown that overexpressed CK1γ synergizes with LRP to activate TCF-dependent transcription. Similar approaches have shown that CK1ε and CK1δ cooperate with the intracellular protein Dishevelled to activate TCF-dependent transcription (reviewed in ref. 5). Importantly, Davidson et al.1 show that CK1γ is necessary for Wnt signaling, as depletion of the Drosophila CK1γ homolog, gish, reduced TCF-dependent transcription1. By contrast, Zeng et al.2 have used dominantnegative mutants to implicate both CK1ε and CK1α as the kinases that phosphorylate residue Thr1493 of LRP6 (Fig. 1b). Previous lossof-function studies support a positive role for CK1ε, called Doubletime in Drosophila, in Wnt signaling9, but they also implicate CK1ε in the maintenance of low basal levels of TCF-dependent transcription10. The intracellular C terminus of the single transmembrane LRP6 molecule has five conserved PPPSP motifs that are required for Wnt signaling and mediate interaction with Axin when phosphorylated11 (reviewed in ref. 12). The new studies1,2 extend these observations by identifying serine and threonine

10

residues that flank the PPPSP motif as CK1 substrates and by showing that phosphorylation of these sites enhances Axin binding (Fig. 1b). Phospho-specific antibodies were used by both groups to specifically identify Thr1479 and Thr1493 as CK1 target sites (Fig. 1b). A cluster of serine residues upstream of the PPPSP motif closest to the N terminus, motif A, are also suggested to be CK1 phosphorylation sites (Fig. 1b, cluster 1). Importantly, phosphorylation of Thr1493 by CK1 requires prephosphorylation of Ser1490 within the PPPSP motif. Phospho-priming at the –3 position is a well-described phenomenon for a subset of CK1 substrates13. Zeng et al.2 suggest that the kinase that primes LRP Thr1493 is GSK-3, another protein whose primary role has been thought to be in promoting β-catenin turnover2,4. In support of this contention, they show that GSK-3 inhibitors and loss of GSK-3 gene function reduce levels of phospho-Thr1490. Zeng et al.2 also show that Wnt activity stimulates Ser1490 (PPPSP) phosphorylation2,11. However, Davidson et al.1 find no evidence for Wnt-dependent phosphorylation of Ser1490, and close examination of data from Zeng et al.2 shows that some Ser1490 phosphorylation remains even after loss of GSK-3 gene function, suggesting that other kinases also target the site. So what does the new data tell us about the mechanism by which Wnt inhibits the activity of the β-catenin turnover complex? The simple model proposed by both groups is that Wnt

Axin β-catenin

GSK

GSK

ligands activate kinases associated with the LRP receptor, leading to phosphorylation of the C terminus and recruitment and inactivation of the Axin-associated β-catenin turnover complex1,2. One of the difficulties in studying this model is that the recruitment of the Axin– β-catenin turnover complex to LRP6 also brings Axin-associated kinases, including CK1 and GSK-3, making it difficult to establish whether LRP6 was phosphorylated before or after the recruitment of Axin. Time-course analyses may help to resolve this issue in the future, although initial studies suggest that Axin recruitment to LRP6 may occur within as little as 2 minutes after ligand addition14. Davidson et al.1 show that CK1γ is localized at the plasma membrane and associates with LRP independently of Axin1. However, they find no evidence for Wnt-regulated changes in CK1γ activity. It thus remains to be determined how liganddependent alterations to LRP phosphorylation (or dephosphorylation) are initiated. Although the new studies have primarily focused on LRP6, the Wnt-bound signaling complex most probably contains the Wnt receptor Frizzled and other intracellular signaling proteins, including Dishevelled, which interacts with the C terminus of Frizzled15. The ligand-dependent association between Frizzled and LRP could bring LRP into proximity with Dishevelled-associated kinases and phosphatases, altering LRP phosphorylation16,17. The subsequent recruitment of Axin-associated kinases may enhance the affinity and stability of

VOLUME 13 NUMBER 1 JANURARY 2006 NATURE STRUCTURAL & MOLECULAR BIOLOGY

© 2006 Nature Publishing Group http://www.nature.com/nsmb

NEWS AND VIEWS an Axin–LRP complex through increased phosphorylation and avidity involving the presence of the multiple Axin-binding motifs (Fig. 2). The tightly bound Axin–LRP complex may be the site at which β-catenin turnover is extinguished, perhaps through processes involving endocytosis or titration of GSK-3 from Axin18–20. In this model, the binding of Axin to LRP would initiate an irreversible process of ever-increasing phosphorylation leading to the recruitment of the endocytic machinery or the titration of GSK-3 from Axin by Frizzled receptor–associated G proteins20. This process could be initiated by a threshold level of LRP phosphorylation. In this context, the identification of CK1γ as an essential but unregulated component may reflect its role in establishing a threshold of CK1 activity at the membrane. So is there any evidence for a Dishevelledassociated kinase whose activity is regulated by Wnt ligands? Again, a CK1 isoform steps forward. Swiatek et al. have shown that CK1ε activity is upregulated by Wnt ligands after

dephosphorylation of its autoinhibitory C terminus21. The enhancement of LRP phosphorylation by Dishevelled-associated kinases may also explain the role of Dishevelled in recruiting Axin to the plasma membrane during signaling22. The further analysis of early Wnt signaling will be a difficult undertaking because of the dynamic positive and negative roles of key components. A useful analogy may be to regard the β-catenin turnover complex as a molecular machine in which kinases such as GSK-3 and CK1 function as cogs in several processes. The trick required to understand the machine’s function may be the removal of selected cogs at defined times. This will require a fine set of molecular tools. 1. Davidson, G. et al. Nature 438, 867–872 (2005). 2. Zeng, X. et al. Nature 438, 873–877 (2005). 3. Schwarz-Romond, T. et al. Genes Dev. 16, 2073– 2084 (2002). 4. Logan, C.Y. & Nusse, R. Annu. Rev. Cell Dev. Biol. 20, 781–810 (2004). 5. Ding, Y. & Dale, T. Trends Biochem. Sci. 27, 327– 329 (2002).

6. Knippschild, U. et al. Cell. Signal. 17, 675–689 (2005). 7. Liu, C. et al. Cell 108, 837–847 (2002). 8. Amit, S. et al. Genes Dev. 16, 1066–1076 (2002). 9. Yanagawa, S. et al. EMBO J. 21, 1733–1742 (2002). 10. Cong, F., Schweizer, L. & Varmus, H. Mol. Cell. Biol. 24, 2000–2011 (2004). 11. Tamai, K. et al. Mol. Cell 13, 149–156 (2004). 12. He, X., Semenov, M., Tamai, K. & Zeng, X. Development 131, 1663–1677 (2004). 13. Marin, O. et al. Proc. Natl. Acad. Sci. USA 100, 10193–10200 (2003). 14. Mao, J. et al. Mol. Cell 7, 801–809 (2001). 15. Wong, H.C. et al. Mol. Cell 12, 1251–1260 (2003). 16. Tamai, K. et al. Nature 407, 530–535 (2000). 17. Cong, F. & Varmus, H. Development 131, 5103– 5108 (2004). 18. Chen, W. et al. Science 301, 1391–1394 (2003). 19. Castellone, M.D., Teramoto, H., Williams, B.O., Druey, K.M. & Gutkind, J.S. Science 310, 1504– 1510 (2005). 20. Liu, X., Rubin, J.S. & Kimmel, A.R. Curr. Biol. 15, 1989–1997 (2005). 21. Swiatek, W. et al. J. Biol. Chem. 279, 13011–13017 (2004). 22. Cliffe, A., Hamada, F. & Bienz, M. Curr. Biol. 13, 960–966 (2003).

Spying on the inner life of a cell Life isn’t simple. And neither is gene activity. The stages involved in expressing a protein are numerous, and because each step is governed by probability, the levels of mRNA and protein within a cell are subject to great variability. Yet the cell must perform certain critical functions and respond appropriately to external stimuli, despite fluctuation in the levels of factors essential for the response. Consequently, studies of the kinetics of gene expression at the singlecell level are necessary to help elucidate how cells function in a noisy environment. Studies of gene expression have suggested a strong distinction between prokaryotes and eukaryotes with regard to its regulation. Data from bacteria suggest that a gene expresses relatively small numbers of mRNAs and that translation occurs in bursts. In eukaryotes, which have the added complexity of a highly organized chromatin structure, the ability to open chromatin for bursts of transcription seems to be more important in terms of overall expression. Previous studies were able to measure protein levels precisely, but mRNA levels could only be estimated. What was needed was a way in which the limiting factor, mRNA, could be directly measured, and this is accomplished in a new study by Golding et al. (Cell 123, 1025–1036, 2005). The EscHerichia coli system they developed has two components: an RNA-binding protein, MS2, fused to GFP and the RFP gene fused to 96 tandem copies of the MS2-binding site. Thus, with appropriate normalization, the focal green signal can be directly related to the level of mRNA, whereas the diffuse red signal corresponds to the protein level. As

shown in the figure, each cell shows different levels of both mRNA and protein that can be quantified. The first conclusion of these studies was that, in fact, expression in bacteria at the single-cell level is more similar to previous eukaryotic data: mRNA expression occurs in intense bursts, which synthesize a random number of transcripts. This is an approximately Poisson process (that is, each burst of transcription produces a geometric distribution of mRNAs). What happens to the mRNAs when the cell divides? Here the data fit exactly to a binomial distribution, providing direct proof that partitioning is random, as expected. The correlation between transcript and protein levels was determined. It has been assumed that there would be proportionality between the two, and this is observed in older cells. In newly divided cells, however, the correlation is significantly weaker. Golding et al. propose that this results from the randomization of mRNAs during division. Protein levels, being high, are distributed virtually equally in the two daughter cells, but transcripts, being fewer in number, are less likely to segregate equally; this magnifies differences in the two cells. As the cell grows, however, this effect is neutralized by protein expression. On average, each mRNA produces 60–110 protein molecules. This work on the kinetics of gene expression in single cells provides novel insight into a stochastic process at a resolution that was previously unattainable. The results indicate that our views about chromatin remodeling as a rate-limiting step in gene expression may need to be reconsidered, and further experiments are required to understand the nature of the burst process.

NATURE STRUCTURAL & MOLECULAR BIOLOGY VOLUME 13 NUMBER 1 JANURARY 2006

Angela K Eggleston

11