The Ezh2 methyltransferase complex: actin up in the cytosol

Update

514

TRENDS in Cell Biology

Acknowledgements Work in the laboratory of Martin Lowe is supported by an MRC Senior Research Fellowship and grants from the Wellcome Trust and BBSRC.

References 1 Van Lint, J. et al. (2002) Protein kinase D: an intracellular traffic regulator on the move. Trends Cell Biol. 12, 193–200 2 Rykx, A. et al. (2003) Protein kinase D: a family affair. FEBS Lett. 546, 81–86 3 Rozengurt, E. et al. (2005) Protein kinase D signaling. J. Biol. Chem. 280, 13205–13208 4 Baron, C.L. and Malhotra, V. (2002) Role of diacylglycerol in PKD recruitment to the TGN and protein transport to the plasma membrane. Science 295, 325–328 5 Oancea, E. et al. (2003) Mechanism of persistent protein kinase D1 translocation and activation. Dev. Cell 4, 561–574 6 Maeda, Y. et al. (2001) Recruitment of protein kinase D to the trans-Golgi network via the first cysteine-rich domain. EMBO J. 20, 5982–5990 7 Hausser, A. et al. (2002) Structural requirements for localization and activation of protein kinase C mu (PKC mu) at the Golgi compartment. J. Cell Biol. 156, 65–74 8 Jamora, C. et al. (1999) Gbetagamma-mediated regulation of Golgi organization is through the direct activation of protein kinase D. Cell 98, 59–68 9 Liljedahl, M. et al. (2001) Protein kinase D regulates the fission of cell surface destined transport carriers from the trans-Golgi network. Cell 104, 409–420 10 Yeaman, C. et al. (2004) Protein kinase D regulates basolateral membrane protein exit from trans-Golgi network. Nat. Cell Biol. 6, 106–112

Vol.15 No.10 October 2005

11 Diaz Anel, A.M. and Malhotra, V. (2005) PKCeta is required for beta1gamma2/beta3gamma2- and PKD-mediated transport to the cell surface and the organization of the Golgi apparatus.. J. Cell Biol. 169, 83–91 12 Goodnight, J.A. et al. (1995) Immunocytochemical localization of eight protein kinase C isozymes overexpressed in NIH 3T3 fibroblasts. Isoform-specific association with microfilaments, Golgi, endoplasmic reticulum, and nuclear and cell membranes. J. Biol. Chem. 270, 9991–10001 13 Lehel, C. et al. (1995) Protein kinase C epsilon is localized to the Golgi via its zinc-finger domain and modulates Golgi function. Proc. Natl. Acad. Sci. U. S. A. 92, 1406–1410 14 Nishikawa, K. et al. (1998) Association of protein kinase Cmu with type II phosphatidylinositol 4-kinase and type I phosphatidylinositol4-phosphate 5-kinase. J. Biol. Chem. 273, 23126–23133 15 Godi, A. et al. (2004) FAPPs control Golgi-to-cell-surface membrane traffic by binding to ARF and PtdIns(4)P. Nat. Cell Biol. 6, 393–404 16 Carnegie, G.K. et al. (2004) AKAP-Lbc nucleates a protein kinase D activation scaffold. Mol. Cell 15, 889–899 17 Torii, S. et al. (2004) Sef is a spatial regulator for Ras/MAP kinase signaling. Dev. Cell 7, 33–44 18 Bonazzi, M. et al. (2005) CtBP3/BARS drives membrane fission in dynamin-independent transport pathways. Nat. Cell Biol. 7, 570–580 19 Bankaitis, V.A. and Morris, A.J. (2003) Lipids and the exocytotic machinery of eukaryotic cells. Curr. Opin. Cell Biol. 15, 389–395

0962-8924/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tcb.2005.08.001

The Ezh2 methyltransferase complex: actin up in the cytosol Jeffrey C. Nolz1, Timothy S. Gomez1 and Daniel D. Billadeau1,2 1 2

Department of Immunology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA Division of Oncology Research, Mayo Clinic College of Medicine, Rochester, MN 55905, USA

Ezh2, a polycomb group protein, is known to function in histone methylation, thereby regulating gene expression. However, in a recent study by Su et al., the Ezh2-containing complex has been given an additional role in cellular regulation. Cytosolic Ezh2 methyltransferase complexes were shown to associate with Vav1 and control receptorinduced actin polymerization and proliferation in a methylation-dependent manner. Overall, these findings implicate lysine methylation as a posttranslational modification crucial for receptor-mediated signal transduction events.

Introduction Signal transduction requires posttranslational modifications to specifically and precisely transform extracellular stimuli into functional cellular outcomes, such as cytoskeletal, transcriptional and proliferative responses. One Corresponding author: Billadeau, D.D. ([email protected]). Available online 26 August 2005 www.sciencedirect.com

such modification, protein methylation, has been primarily associated with histones, playing an essential role in chromatin remodeling and cell lineage determination/ maintenance during development [1]. Emerging evidence also supports a role for protein methylation in various cellular processes apart from chromatin structure [2–7], but has so far been primarily associated with transcriptional regulation (Table 1). Intriguingly, Su et al. have recently added to our knowledge of this phenomenon by characterizing a role for a cytosolic complex containing Ezh2, a protein with known histone-methyltransferase (HMT) activity, demonstrating that Ezh2 methyltransferase activity is necessary for receptor-mediated signals leading to actin reorganization and proliferation in both T lymphocytes and fibroblasts [8] (Figure 1). An Ezh2 complex in the cytosol Ezh2 is one of the mammalian homologs of Drosophila Enhancer of Zeste E(z), a member of the polycomb group

Update

TRENDS in Cell Biology

Vol.15 No.10 October 2005

515

Table 1. Examples of proteins known to be methylated Protein p53

Type of methylation Lysine

Acting methyltransferase SET9

TAF10

Lysine

SET9

STAT1 p300/CBP NIP45 RNA helicase A

Arginine Arginine Arginine Arginine

PRMT1 CARM1 PRMT1 PRMT1

Putative effect

Refs

Activation/increased affinity for chromatin binding at target genes/ stability Enhanced transcriptional activity from increased affinity for RNA polymerase II Impaired binding to inhibitor protein PIAS1 Inhibits transcriptional activity by blocking interaction with CREB Facilitates interaction with NF-AT leading to augmented gene expression Regulates nuclear import

[2]

of proteins, which form distinct nuclear complexes and function to repress homeobox genes during Drosophila development [9]. Not surprisingly, Ezh2 has also been shown to be a crucial epigenetic regulator of embryonic development in mammals [10]. Su et al. have already

(a)

[3] [4] [5] [6] [7]

identified that Ezh2-deficient B cells selectively lose histone H3 trimethylation at lysine 27 (H3-K27) and do not rearrange immunoglobulin heavy chains during B cell development [11], suggesting a gene-regulatory function in immune cells. Interestingly, they now reveal

(b)

APC PDGF

MHC

CD4

TCR

T cell

Dorsal ruffles

Lck

LAT

ZAP70

Actin reorganization

Actin reorganization

Arp2/3 N-WASp

76

Cdc42

P SL Vav1 Cdc42

Arp2/3

? H3 -C

Cdc42

WASp

GDP

? -CH3 GTP

Ezh2

GDP

EED SUZ12

?

-CH3

GTP Cdc42

SUZ12 Ezh2

Vav2/3

EED

? -CH3 ?

H3

-CH 3

?

?

?

Proliferation

Proliferation

-C

TRENDS in Cell Biology

Figure 1. The role of Ezh2 in the cytosol of T lymphocytes and fibroblasts. (a) T lymphocyte activation involves the recognition of a specific peptide–MHC complex on an antigen-presenting cell (APC), leading to the activation of proximal tyrosine kinases Lck and ZAP-70, which in turn phosphorylate various downstream signaling intermediates, including the adaptors LAT and SLP76. These events lead to the recruitment of Vav1, which, upon activation, facilitates the exchange of GTP for GDP on Rho-family GTPases such as Cdc42 and Rac1. In particular, active GTP-bound Cdc42 can then interact with multiple downstream effectors, such as the Wiskott–Aldrich Syndrome protein (WASp), resulting in actin polymerization and reorganization. Su et al. propose that the cytosolic Ezh2 complex interacts with Vav1 in the cytosol and methylates certain regulatory proteins that control the activation of Cdc42 by active Vav1. These methylated proteins might be directly bound to Vav1, thereby modulating its ability to regulate GTPases or, by contrast, might be affecting the active states of GTPases following Vav1-stimulated exchange. (b) In addition, the authors hypothesize that a similar complex consisting of Ezh2 and Vav2 or Vav3 might form in response to growth factor receptor stimulation in fibroblasts, although it is unknown whether Ezh2 interacts with Vav2/Vav3. Stimulation of the PDGF receptor on fibroblasts results in phosphorylation of Vav2/Vav3, resulting in Rac1 and Cdc42 activation, which might also be dependent upon the methyltransferase activity of the Ezh2 complex. In addition, as shown in (a) and (b), the cytosolic Ezh2 complex might methylate certain proteins involved in signal transduction, leading to cellular proliferation. www.sciencedirect.com

516

Update

TRENDS in Cell Biology

that there is also an unexpected necessity for the methyltransferase activity of Ezh2 outside of the nucleus [8]. It is known that the HMT activity of Ezh2 in the nucleus is dependent on its association with other proteins, such as Suppressor of Zeste 12 (SUZ12) and Embryonic Ectoderm Development (EED) [12], which Su et al. also show associates with cytosolic Ezh2 [8]. In addition to these binding partners, the cytosolic Ezh2 methyltransferase complex in T cells was found to be associated with another previously identified nuclear Ezh2-ineractor, the Rho GDP–GTP exchange factor (GEF) Vav1 [13]. Vav1 is a key signaling component downstream of various receptors, including growth-factor receptors and the T cell receptor (TCR), and is primarily expressed in immune cells, where it is known to function as a scaffold protein and actin regulator through its control of guanine nucleotide exchange on the Rho family of GTPases [14]. Interestingly, Ezh1, the second homolog of Drosophila E(z), has also been shown to be expressed and localized in the cytoplasm of T cell lines and is phosphorylated by Lck, leading to an association with ZAP-70, suggesting that each homolog might have a distinct function in the signaling pathways leading to T cell activation [15]. In T cells, recognition of a specific peptide–MHC complex by the TCR (Figure 1a) initiates the activation of the proximal tyrosine kinases Lck and ZAP-70, leading to LAT phosphorylation and subsequent recruitment of a variety of proteins known to promote actin reorganization, including SLP76, Vav1 and WASp, as well as those involved in signaling cascades leading to cytokine production. Reorganization of actin is believed to be a crucial step in the proper formation of an immunological synapse (IS) between an antigen-presenting cell (APC) and the T cell, and is thought to be crucial for the assembly of signaling complexes leading to T cell activation. While it was shown that actin accumulation at the IS was impaired in Ezh2K/K T cells, feasibly the basis for the decreased proliferation in response to TCR ligation, any specific consequence of the actin defect was not explored. The authors do demonstrate, however, that treatment of Ezh2K/K cells with phorbol myristate acetate (PMA) and an ionophore, which presumably bypasses proximal signaling events and the need for proper actin assembly, rescues the proliferation defect, suggesting that Ezh2 is in fact playing an essential role in receptor-proximal signaling events. Cytosolic Ezh2 also appears to be acting similarly downstream of activated growth-factor receptors, which normally promote dynamic cytoskeletal responses, cell migration, proliferation and survival. Su et al. reveal that Ezh2K/K mouse embryonic fibroblasts lose dorsal circular ruffle formation, which is thought to be important for the disassembly and remodeling of static actin structures during migration, and do not proliferate in response to platelet-derived growth factor (PDGF). In addition, like T cells, the actin dynamics of PDGF-receptor-stimulated fibroblasts also appear to depend on Ezh2-controlled signaling pathways leading to proper GTPase activation (Figure 1b). www.sciencedirect.com

Vol.15 No.10 October 2005

Thus, although the role of the Ezh2–Vav1 interaction remains to be fully elucidated, Su et al. demonstrate a requirement for Vav1 downstream of Ezh2 in TCR-stimulated cellular responses (Figure 1a) and, furthermore, propose that the two structurally related and ubiquitously expressed Vav proteins, Vav2 and Vav3, might be important for cytosolic Ezh2 function in fibroblasts (Figure 1b). The significance of a Vav–Ezh2 interaction Ezh2 binds to amino acids 66–115 of Vav1, encompassing the C-terminal half of the Vav1 calponin-homology (CH) domain [13], which has been previously implicated in the auto-inhibition of Vav1, as well as regulating TCR-mediated calcium flux in hematopoietic cells [14]. Interestingly, an oncogenic version of Vav1 (D1–65) rescues actin-related defects in dorsal circular membrane ruffling of Ezh2K/K fibroblasts. However, it remains to be determined whether binding of the Ezh2 complex to Vav1 modulates the GEF activity of Vav1 by stabilizing the active conformation or, by contrast, whether Vav1 recruits the Ezh2 complex to sites of receptor activation, where it can then modify and possibly activate proteins through lysine-methylation. In response to TCR ligation, Vav1 becomes phosphorylated within its acidic region on Tyr174, which releases auto-inhibition, thus activating GEF function [14]. Although Ezh2K/K T cells had diminished activation of the Vav1 target Cdc42, phosphorylation at Tyr174 in Vav1 was unaffected in Ezh2K/K T cells, which might suggest that the Vav1 GEF activity is not impaired in the absence of the Ezh2 complex. A role for the methyltransferase activity of Ezh2 in this process remains a mystery, as the authors confess that they were unable to detect lysine methylation of Vav1 itself. Therefore, they propose that methylation of certain Vav1-associated proteins might be required for the assembly of a proper complex leading to efficient exchange on GTPases (Figure 1). Regardless, these experiments point to lysine methylation by the Ezh2 complex as a key event in actin reorganization and eventual T cell activation and suggest that Ezh2 is required for GDP–GTP exchange upon Rho GTPases by Vav1. While impaired actin reorganization and proliferation in peripheral Ezh2K/K T cells seemed to depend primarily on a defect in signaling pathways, the same did not necessarily hold true for Ezh2K/K T cell development, in which there appeared to be a global decrease in H3-K27 trimethylation in double-negative (DN) thymocytes. The transition from DN (CD4K/CD8K) to double-positive (DP) (CD4C/CD8C) thymocytes is dependent on the ability for signaling events to occur through the pre-TCR that lead to differential gene regulation and proliferation. The DP stage is reached after proliferation ceases, CD4 and CD8 co-receptors are expressed, and the a-chain locus of the TCR has undergone proper rearrangement. Vav1K/K thymocytes are also blocked at the DN stage [14]. Therefore, while it remains plausible that impaired Vav1 function might be the mechanism by which Ezh2K/K T cells are blocked at the DN stage, one cannot completely disregard the histone-methylating activity of the Ezh2

Update

TRENDS in Cell Biology

complex in thymocyte development. In contrast to B cells, however, there appears to be no defect in the ability to properly rearrange the TCR [11]. To further complicate matters, recent evidence has also suggested that Vav1 can translocate to the nucleus and participate in transcriptional regulation of NF-AT and NFkB target genes [16], thus raising the possibility that Vav1 also participates in the modulation of gene transcription following cellular activation, through the associated Ezh2 methyltransferase activity. However, the ability of the Vav triple knockout (Vav1–Vav3) mouse to develop normally disputes a role for Vav proteins in the coordination of histone-directed methylation by the Ezh2 complex [17]. However, the question can be raised as to whether this interaction is important for gene regulation in developing thymocytes or in other cell types following receptor engagement. Concluding remarks In their recent article, Su et al. have discovered a role for Ezh2 as a crucial regulator of actin cytoskeletal dynamics and proliferative responses downstream of activated cellsurface receptors, suggesting an essential role for lysine methylation in receptor-mediated signal transduction events. This exciting finding bolsters the notion that protein methylation is commonplace in signal transduction, perhaps resulting in distinct functional consequences, and adds to the complexity of protein regulatory mechanisms. Su et al. provide a novel means by which receptorproximal protein methylation could occur during ligandinduced cellular responses. Although the exact significance of the Vav1–Ezh2 interaction is not entirely clear, and potential substrates of the cytosolic Ezh2 complex have yet to be identified, the idea that protein methylation plays a role in signal transduction is attractive. Therefore, identification of the methylated targets of the Ezh2 complex following receptor stimulation will enhance our understanding of such novel modes of protein regulation and will further illuminate the subtle complexities of protein structure and function.

Vol.15 No.10 October 2005

517

References 1 Sims, R.J., 3rd. et al. (2003) Histone lysine methylation: a signature for chromatin function. Trends Genet. 19, 629–639 2 Chuikov, S. et al. (2004) Regulation of p53 activity through lysine methylation. Nature 432, 353–360 3 Kouskouti, A. et al. (2004) Gene-specific modulation of TAF10 function by SET9-mediated methylation. Mol. Cell 14, 175–182 4 Mowen, K.A. et al. (2001) Arginine methylation of STAT1 modulates IFNalpha/beta-induced transcription. Cell 104, 731–741 5 Xu, W. et al. (2001) A transcriptional switch mediated by cofactor methylation. Science 294, 2507–2511 6 Mowen, K.A. et al. (2004) Arginine methylation of NIP45 modulates cytokine gene expression in effector T lymphocytes. Mol. Cell 15, 559–571 7 Smith, W.A. et al. (2004) Arginine methylation of RNA helicase a determines its subcellular localization. J. Biol. Chem. 279, 22795–22798 8 Su, I.H. et al. (2005) Polycomb group protein ezh2 controls actin polymerization and cell signaling. Cell 121, 425–436 9 Ringrose, L. and Paro, R. (2004) Epigenetic regulation of cellular memory by the Polycomb and Trithorax group proteins. Annu. Rev. Genet. 38, 413–443 10 O’Carroll, D. et al. (2001) The polycomb-group gene Ezh2 is required for early mouse development. Mol. Cell. Biol. 21, 4330–4336 11 Su, I.H. et al. (2003) Ezh2 controls B cell development through histone H3 methylation and Igh rearrangement. Nat. Immunol. 4, 124–131 12 Cao, R. and Zhang, Y. (2004) SUZ12 is required for both the histone methyltransferase activity and the silencing function of the EED-EZH2 complex. Mol. Cell 15, 57–67 13 Hobert, O. et al. (1996) Interaction of Vav with ENX-1, a putative transcriptional regulator of homeobox gene expression. Mol. Cell. Biol. 16, 3066–3073 14 Turner, M. and Billadeau, D.D. (2002) VAV proteins as signal integrators for multi-subunit immune-recognition receptors. Nat. Rev. Immunol. 2, 476–486 15 Ogawa, M. et al. (2003) The Polycomb-group protein ENX-2 interacts with ZAP-70. Immunol. Lett. 86, 57–61 16 Houlard, M. et al. (2002) Vav1 is a component of transcriptionally active complexes. J. Exp. Med. 195, 1115–1127 17 Fujikawa, K. et al. (2003) Vav1/2/3-null mice define an essential role for Vav family proteins in lymphocyte development and activation but a differential requirement in MAPK signaling in T and B cells. J. Exp. Med. 198, 1595–1608

0962-8924/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tcb.2005.08.003

Erratum

Erratum: Midbodies and phragmoplasts: analogous structures involved in cytokinesis Trends Cell Biology 15 (2005), 404–413

In this article by Ahna R. Skop and colleagues, which was published in the August 2005 issue of TCB, there was a production error that resulted in the omission of a footnote indicating that Marisa S. Otegui and Koen J. Verbrugghe contributed equally to the article and were

DOI of original article: 10.1016/j.tcb.2005.06.003 www.sciencedirect.com

therefore co-first-authors. We apologize to the authors for this error. 0962-8924/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tcb.2005.08.008