Actin Post-translational Modifications: The Cinderella of Cytoskeletal Control

Post-translational modifications of actin affect its folding and structure, as well as interaction with actin-binding proteins, and thus interfere with cytoskeleton dynamics. The actin N-terminal acetyltransferase, NAA80, was recently identified, thus solving a 30-year-old mystery on the final step of actin's unique and conserved N-terminal maturation process. Acetylation and arginylation compete for actin's N terminus, both affecting filament formation, interaction with actin-binding proteins, and cell motility. Actin oxidation of Met44 and Met47 by the MICAL enzymes promotes, in synergy with cofilin, the disassembly of actin filaments and is linked to cancer development. Toxin-mediated modifications of actin may lead to actin filament aggregation, and in some cases cell death. Actin is one of the most abundant proteins in eukaryotic cells and the main component of the microfilament system. It plays essential roles in numerous cellular activities, including muscle contraction, maintenance of cell integrity, and motility, as well as transcriptional regulation. Besides interacting with various actin-binding proteins (ABPs), proper actin function is regulated by post-translational modifications (PTMs), such as acetylation, arginylation, oxidation, and others. Here, we explain how actin PTMs can contribute to filament formation and stability, and may have additional actin regulatory functions, which potentially contribute to disease development. Actin is one of the most abundant proteins in eukaryotic cells and the main component of the microfilament system. It plays essential roles in numerous cellular activities, including muscle contraction, maintenance of cell integrity, and motility, as well as transcriptional regulation. Besides interacting with various actin-binding proteins (ABPs), proper actin function is regulated by post-translational modifications (PTMs), such as acetylation, arginylation, oxidation, and others. Here, we explain how actin PTMs can contribute to filament formation and stability, and may have additional actin regulatory functions, which potentially contribute to disease development. Actin (see Glossary) accounts for up to ∼15% of the total protein level in muscle cells and 1–3% in nonmuscle cells. It exists in both a monomeric globular state (G-actin) and polymerized filamentous state (F-actin; Figure 1A), and the switch between the two states is highly dynamic. The actin filaments play crucial roles in countless cellular functions, including muscle contraction, cell signaling, as well as cell integrity and motility [1Pollard T.D. Actin and actin-binding proteins.Cold Spring Harb. Perspect. Biol. 2016; 8a018226Crossref PubMed Scopus (366) Google Scholar]. The multifunctionality of actin is based on three pillars (Figure 1B): chaperonin-assisted folding [2Balchin D. et al.Pathway of actin folding directed by the eukaryotic chaperonin TRiC.Cell. 2018; 174: 1507-1521Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar], interactions with actin-binding proteins (ABPs) [1Pollard T.D. Actin and actin-binding proteins.Cold Spring Harb. Perspect. Biol. 2016; 8a018226Crossref PubMed Scopus (366) Google Scholar], and post-translational modifications (PTMs; Figure 1C) [3Terman J.R. Kashina A. Post-translational modification and regulation of actin.Curr. Opin. Cell Biol. 2013; 25: 30-38Crossref PubMed Scopus (148) Google Scholar]. Numerous studies and reviews describe the influence ABPs have on the actin cytoskeleton. In this review, however, we describe the most recent findings on actin PTMs shedding light on a crucial, but often overlooked, aspect of actin biology. Actins represent a family of isoforms which are highly similar in sequence (≥93% sequence identity) and each conserved throughout evolution. Based on their amino acid sequences, six isoforms were described and classified according to the tissues in which they were found in mammals and birds: four muscle forms; α-skeletal, α-cardiac, α-smooth, γ-smooth, and two nonmuscle cytoplasmic actins: β-cytoplasmic and γ-cytoplasmic [4Vandekerckhove J. Weber K. At least six different actins are expressed in a higher mammal: analysis based on the amino-acid sequence of the amino-terminal tryptic peptide.J. Mol. Biol. 1978; 126: 783-802Crossref PubMed Scopus (532) Google Scholar]. α, β, and γ refer to their respective mobility during isoelectric focusing, which is exclusively due to the number (3/4) and nature (Asp/Glu) of the N-terminal acidic residues. For example, the N terminus of β-cytoplasmic actin is Ac-DDDIAALVV- while that of γ-cytoplasmic actin is Ac-EEEIAALVI-. The four underlined residues constitute the only differences in a total of 375 residues present in these two isoforms, emphasizing their conserved nature. Despite their sequence and structural similarities, actin isoforms display both overlapping and unique cellular roles (reviewed in [5Perrin B.J. Ervasti J.M. The actin gene family: function follows isoform.Cytoskeleton. 2010; 67: 630-634Crossref Scopus (219) Google Scholar]). This has been clearly demonstrated in mice where knockout of β-actin results in embryonic lethality [6Shawlot W. et al.Restricted β-galactosidase expression of a hygromycin-lacZ gene targeted to the β-actin locus and embryonic lethality of β-actin mutant mice.Transgenic Res. 1998; 7: 95-103Crossref PubMed Scopus (77) Google Scholar, 7Shmerling D. et al.Strong and ubiquitous expression of transgenes targeted into the β-actin locus by Cre/lox cassette replacement.Genesis. 2005; 42: 229-235Crossref PubMed Scopus (65) Google Scholar], while γ-actin-deficient mice show developmental defects, but are viable [8Belyantseva I.A. et al.γ-Actin is required for cytoskeletal maintenance but not development.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 9703-9708Crossref PubMed Scopus (119) Google Scholar, 9Bunnell T.M. Ervasti J.M. Delayed embryonic development and impaired cell growth and survival in Actg1 null mice.Cytoskeleton. 2010; 67: 564-572Crossref Scopus (60) Google Scholar]. Although these remarkably different effects are not yet fully understood, it is known that these two isoactins display distinct intracellular localization patterns [5Perrin B.J. Ervasti J.M. The actin gene family: function follows isoform.Cytoskeleton. 2010; 67: 630-634Crossref Scopus (219) Google Scholar]. Further in vitro experiments reveal that mixtures of isoactins in filaments could affect polymerization dynamics, stability, and interactions with ABPs [5Perrin B.J. Ervasti J.M. The actin gene family: function follows isoform.Cytoskeleton. 2010; 67: 630-634Crossref Scopus (219) Google Scholar, 10Bergeron S.E. et al.Ion-dependent polymerization differences between mammalian β- and γ-nonmuscle actin isoforms.J. Biol. Chem. 2010; 285: 16087-16095Crossref PubMed Scopus (84) Google Scholar]. On top of these subtle differences, PTMs could contribute by affecting actin structure, localization, and function. Most PTMs will affect the isoactins in a similar manner, given the actin sequence similarities. However, as described later in this review, there are clear cases of isoform-specific PTMs contributing to differentiated functions. The first actin PTM, N-terminal (Nt) acetylation, was reported for skeletal muscle actin in 1966 by Gaetjens and Bárány [11Gaetjens E. Bárány M. N-acetylaspartic acid in G-actin.Biochim. Biophys. Acta. 1966; 117: 176-183Crossref PubMed Scopus (26) Google Scholar], and later identified in all other actin isoforms. Today, more than 140 PTMs have been described in eukaryotic actin sequences ([3Terman J.R. Kashina A. Post-translational modification and regulation of actin.Curr. Opin. Cell Biol. 2013; 25: 30-38Crossref PubMed Scopus (148) Google Scholar] and http://www.phosphosite.org). Some actin PTMs are quantitative and reversible, whereas others are rare, affecting only a minority of the molecules that make up the cellular actin pool. Thus, many actin PTMs should be considered as partial modifications. Actin PTMs are found on 94 different side chains (Table 1, Key Table) which constitute about 45% of the residues that can be modified. Specifically, new phosphorylation, ubiquitination, and SUMOylation sites have been identified by global proteomics analyses in recent years [12Mertins P. et al.Ischemia in tumors induces early and sustained phosphorylation changes in stress kinase pathways but does not affect global protein levels.Mol. Cell. Proteomics. 2014; 13: 1690-1704Crossref PubMed Scopus (257) Google Scholar, 13Mertins P. et al.Proteogenomics connects somatic mutations to signalling in breast cancer.Nature. 2016; 534: 55-62Crossref PubMed Scopus (978) Google Scholar, 14Tsai C.F. et al.Large-scale determination of absolute phosphorylation stoichiometries in human cells by motif-targeting quantitative proteomics.Nat. Commun. 2015; 6: 6622Crossref PubMed Scopus (119) Google Scholar, 15Lumpkin R.J. et al.Site-specific identification and quantitation of endogenous SUMO modifications under native conditions.Nat. Commun. 2017; 8: 1171Crossref PubMed Scopus (67) Google Scholar, 16Lundby A. et al.Proteomic analysis of lysine acetylation sites in rat tissues reveals organ specificity and subcellular patterns.Cell Rep. 2012; 2: 419-431Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar, 17Hendriks I.A. et al.Uncovering global SUMOylation signaling networks in a site-specific manner.Nat. Struct. Mol. Biol. 2014; 21: 927-936Crossref PubMed Scopus (336) Google Scholar, 18Impens F. et al.Mapping of SUMO sites and analysis of SUMOylation changes induced by external stimuli.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 12432-12437Crossref PubMed Scopus (113) Google Scholar, 19Rolland D. et al.Global phosphoproteomic profiling reveals distinct signatures in B-cell non-Hodgkin lymphomas.Am. J. Pathol. 2014; 184: 1331-1342Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar]. Interestingly, we have noticed regions where the frequency of PTMs is significantly lower than average (regions: 95–145, 240–256, and 331–354). This follows the overall accessibility of the side chain residues in the actin structure, though loss of ATP/ADP or internal cleavages could also induce partial denaturation, resulting in unspecific low-level modifications. It is currently not clear to which extent the latter contribute to actin's cellular role, or whether they should be considered as structural noise. Furthermore, our knowledge about the regulation, reversibility, and the interplay between individual PTMs remains limited. Given the high number of reported actin PTMs and the absence of detailed studies for most of them, we focus here predominantly on recent reports covering Nt-acetylation, Nt-arginylation, and oxidation of actin. We discuss their molecular and physiological consequences, and their potential role in disease development.Table 1Key TableActin PTMModified residuesaHighlighted in bold: amino acid modifications described in this review.AcetylationMet1, Asp2, Glu2, Cys2dModified residues that are observed in class II actins (α-cardiac, α-smooth, α-skeletal, and γ-smooth) where the N terminus starts with MC-., Asp3dModified residues that are observed in class II actins (α-cardiac, α-smooth, α-skeletal, and γ-smooth) where the N terminus starts with MC-., Lys50bAmino acid resides known to be modified by two or more PTMs., Lys52dModified residues that are observed in class II actins (α-cardiac, α-smooth, α-skeletal, and γ-smooth) where the N terminus starts with MC-., Lys61, Lys68, Lys113bAmino acid resides known to be modified by two or more PTMs., Lys191bAmino acid resides known to be modified by two or more PTMs., Lys193dModified residues that are observed in class II actins (α-cardiac, α-smooth, α-skeletal, and γ-smooth) where the N terminus starts with MC-., Lys213bAmino acid resides known to be modified by two or more PTMs., Lys315bAmino acid resides known to be modified by two or more PTMs., Lys326bAmino acid resides known to be modified by two or more PTMs., Lys328bAmino acid resides known to be modified by two or more PTMs.ADP-ribosylationArg28cOnly described in non-mammalian actins., Arg95cOnly described in non-mammalian actins., Thr148bAmino acid resides known to be modified by two or more PTMs., Arg177bAmino acid resides known to be modified by two or more PTMs., Arg206cOnly described in non-mammalian actins., Arg372cOnly described in non-mammalian actins.ArginylationAsp3, Ser52bAmino acid resides known to be modified by two or more PTMs., Ser54dModified residues that are observed in class II actins (α-cardiac, α-smooth, α-skeletal, and γ-smooth) where the N terminus starts with MC-., Ile87bAmino acid resides known to be modified by two or more PTMs., dModified residues that are observed in class II actins (α-cardiac, α-smooth, α-skeletal, and γ-smooth) where the N terminus starts with MC-., Phe90, Gly152dModified residues that are observed in class II actins (α-cardiac, α-smooth, α-skeletal, and γ-smooth) where the N terminus starts with MC-., Leu295dModified residues that are observed in class II actins (α-cardiac, α-smooth, α-skeletal, and γ-smooth) where the N terminus starts with MC-., Asn299bAmino acid resides known to be modified by two or more PTMs., dModified residues that are observed in class II actins (α-cardiac, α-smooth, α-skeletal, and γ-smooth) where the N terminus starts with MC-.CarbonylationHis40, His87bAmino acid resides known to be modified by two or more PTMs., His173, Cys374bAmino acid resides known to be modified by two or more PTMs.CrosslinkingLys50/Glu270bAmino acid resides known to be modified by two or more PTMs.Disulfide bondCys285bAmino acid resides known to be modified by two or more PTMs., Cys374bAmino acid resides known to be modified by two or more PTMs.GlutathionylationCys217bAmino acid resides known to be modified by two or more PTMs., Cys374bAmino acid resides known to be modified by two or more PTMs.MethylationLys18bAmino acid resides known to be modified by two or more PTMs., Lys68bAmino acid resides known to be modified by two or more PTMs., His73bAmino acid resides known to be modified by two or more PTMs., Lys84, Ile87bAmino acid resides known to be modified by two or more PTMs., dModified residues that are observed in class II actins (α-cardiac, α-smooth, α-skeletal, and γ-smooth) where the N terminus starts with MC-., Asn299bAmino acid resides known to be modified by two or more PTMs., dModified residues that are observed in class II actins (α-cardiac, α-smooth, α-skeletal, and γ-smooth) where the N terminus starts with MC-., Lys326bAmino acid resides known to be modified by two or more PTMs., cOnly described in non-mammalian actins.Tyrosine nitrationTyr53bAmino acid resides known to be modified by two or more PTMs., Tyr69bAmino acid resides known to be modified by two or more PTMs., Tyr91bAmino acid resides known to be modified by two or more PTMs., Tyr198bAmino acid resides known to be modified by two or more PTMs., Tyr218bAmino acid resides known to be modified by two or more PTMs., Tyr240bAmino acid resides known to be modified by two or more PTMs., Tyr294bAmino acid resides known to be modified by two or more PTMs., Tyr362bAmino acid resides known to be modified by two or more PTMs.S-nitrosylationCys217bAmino acid resides known to be modified by two or more PTMs., Cys257bAmino acid resides known to be modified by two or more PTMs., Cys285bAmino acid resides known to be modified by two or more PTMs., Cys374bAmino acid resides known to be modified by two or more PTMs.OxidationCys17, Met44, Met47, Trp81dModified residues that are observed in class II actins (α-cardiac, α-smooth, α-skeletal, and γ-smooth) where the N terminus starts with MC-., Met82, Trp88dModified residues that are observed in class II actins (α-cardiac, α-smooth, α-skeletal, and γ-smooth) where the N terminus starts with MC-., Met178, Met190, Cys217bAmino acid resides known to be modified by two or more PTMs., Met227, Cys257bAmino acid resides known to be modified by two or more PTMs., Met269, Cys272bAmino acid resides known to be modified by two or more PTMs., Cys285bAmino acid resides known to be modified by two or more PTMs., Met235, Trp342dModified residues that are observed in class II actins (α-cardiac, α-smooth, α-skeletal, and γ-smooth) where the N terminus starts with MC-., Met355, Trp358dModified residues that are observed in class II actins (α-cardiac, α-smooth, α-skeletal, and γ-smooth) where the N terminus starts with MC-., Cys374bAmino acid resides known to be modified by two or more PTMs.PhosphorylationSer14, Ser33, Ser52bAmino acid resides known to be modified by two or more PTMs., Tyr53bAmino acid resides known to be modified by two or more PTMs., Ser60, Thr66, Tyr69bAmino acid resides known to be modified by two or more PTMs., Thr77, Thr89, Tyr91bAmino acid resides known to be modified by two or more PTMs., Tyr143, Thr148bAmino acid resides known to be modified by two or more PTMs., S155, Thr160, Thr162, Tyr166, Tyr169, Thr186, Tyr198bAmino acid resides known to be modified by two or more PTMs., Ser199bAmino acid resides known to be modified by two or more PTMs., Thr201, Ser201dModified residues that are observed in class II actins (α-cardiac, α-smooth, α-skeletal, and γ-smooth) where the N terminus starts with MC-., Thr202, Thr203, Tyr218bAmino acid resides known to be modified by two or more PTMs., Thr229, Ser233, S235, Ser239, Tyr240bAmino acid resides known to be modified by two or more PTMs., Thr249, Thr262dModified residues that are observed in class II actins (α-cardiac, α-smooth, α-skeletal, and γ-smooth) where the N terminus starts with MC-., S265, S271, Tyr294bAmino acid resides known to be modified by two or more PTMs., Thr297, S300, Tyr306, Thr318, Ser323, Thr324, Ser324cOnly described in non-mammalian actins. Tyr362bAmino acid resides known to be modified by two or more PTMs., Ser365SUMOylationLys61bAmino acid resides known to be modified by two or more PTMs., Lys68bAmino acid resides known to be modified by two or more PTMs., Lys84bAmino acid resides known to be modified by two or more PTMs., Lys113bAmino acid resides known to be modified by two or more PTMs., Lys284bAmino acid resides known to be modified by two or more PTMs., Lys291bAmino acid resides known to be modified by two or more PTMs., Lys315bAmino acid resides known to be modified by two or more PTMs., Lys326bAmino acid resides known to be modified by two or more PTMs., Lys328bAmino acid resides known to be modified by two or more PTMs.UbiquitinationLys18bAmino acid resides known to be modified by two or more PTMs., Lys50bAmino acid resides known to be modified by two or more PTMs., Lys61bAmino acid resides known to be modified by two or more PTMs., Lys68bAmino acid resides known to be modified by two or more PTMs., Lys84bAmino acid resides known to be modified by two or more PTMs., Lys113bAmino acid resides known to be modified by two or more PTMs., Lys118cOnly described in non-mammalian actins. Lys191bAmino acid resides known to be modified by two or more PTMs., Lys213bAmino acid resides known to be modified by two or more PTMs., Lys215, Lys238, Lys284bAmino acid resides known to be modified by two or more PTMs., Lys291bAmino acid resides known to be modified by two or more PTMs., Lys315bAmino acid resides known to be modified by two or more PTMs., Lys326bAmino acid resides known to be modified by two or more PTMs., Lys328bAmino acid resides known to be modified by two or more PTMs., Lys359a Highlighted in bold: amino acid modifications described in this review.b Amino acid resides known to be modified by two or more PTMs.c Only described in non-mammalian actins.d Modified residues that are observed in class II actins (α-cardiac, α-smooth, α-skeletal, and γ-smooth) where the N terminus starts with MC-. Open table in a new tab Although not all actin PTMs appear at the same time on the same molecule, and some PTMs have only been reported in particular organisms, their sheer number poses a serious challenge for a global understanding of their regulatory mechanisms. For instance, how can an actin molecule, whose primary role is to generate dynamic filaments composed of geometrically conserved building blocks repeated over several thousand times, give rise to these structures when decorated with potentially structure disturbing PTMs? How can both G- and F-actin interact in a dynamic and rigorously controlled manner with a plethora of ABPs when carrying this large number of modifications? PTMs can however participate in the structural architecture of actin and modify their filaments. One of the best known examples is the structure of arthrin, a 55-kDa heavy form of actin first observed in insect muscle thin filaments [20Bullard B. et al.Arthrin: a new actin-like protein in insect flight muscle.J. Mol. Biol. 1985; 182: 443-454Crossref PubMed Scopus (37) Google Scholar]. This insect actin, which is monoubiquitinated at Lys118 (Table 1), appears at every seventh subunit along the filament long pitch helices. It was suggested that arthrin regulates muscle contractile activity [20Bullard B. et al.Arthrin: a new actin-like protein in insect flight muscle.J. Mol. Biol. 1985; 182: 443-454Crossref PubMed Scopus (37) Google Scholar]. A more recent report on structural regulation of the actin filament network refers to Nt-arginylation of β-actin by arginyl-tRNA protein transferase 1 (ATE1). In this case, Nt-arginylated actins form normal filament structures. Non-Nt-arginylated actin isolated from ATE1 knockout (KO) cells, on the other hand, forms bundles and aggregates, resulting in shorter filaments. On a cellular level this leads to disorganization of lamellipodia and filopodia, an effect which is attributed to altered interactions with ABPs [21Saha S. et al.Arginylation regulates intracellular actin polymer level by modulating actin properties and binding of capping and severing proteins.Mol. Biol. Cell. 2010; 21: 1350-1361Crossref PubMed Scopus (63) Google Scholar]. Given the multifunctional nature of actin, one can expect that the final outcome of this high number of PTMs could be extremely complex. Some PTMs will affect steady-state filament growth by blocking one of the filament ends or reducing the concentration of polymerization competent monomers. Some PTMs may interfere with the actin-ABP equilibrium or drive actin molecules towards degradation pathways. And if this is not yet sufficiently complex, PTMs may enhance or switch off each other's effects by crosstalking mechanisms. The circuits that are produced could function via loops that on their turn activate novel circuits. These quantum bits of modifications are most likely not simply noise, but could push the cell following stochastic mechanisms towards a reversible or irreversible destiny. For instance, Tyr53 can be a target for phosphorylation, but also for nitration during oxidative stress. Similarly, Cys374 is highly reactive and can accept different types of modifications (Table 1). It is not clear whether these modifications will result in the same effect because they display a different chemical nature. An interesting example of the complexity involves some prominent ABPs like ADF/cofilin, gelsolin (Figure 1D), profilin, and DNase I (Figure 1E). Profilin binds to two regions in actin (Figure 1E), while cofilin interacts with actin via three sites (Figure 1D) [1Pollard T.D. Actin and actin-binding proteins.Cold Spring Harb. Perspect. Biol. 2016; 8a018226Crossref PubMed Scopus (366) Google Scholar, 22Tanaka K. et al.Structural basis for cofilin binding and actin filament disassembly.Nat. Commun. 2018; 9: 1860Crossref PubMed Scopus (91) Google Scholar]. Part of these sites overlap with each other. Thus, modifications in actin could tilt the balance by which these two ABPs exert their control on actin assembly. N-terminal maturation of actin is an elegant example where a particular protein modification depends on the previous one. Here, the successive actions of methionine aminopeptidases, N-terminal acetyltransferases, and ATE1 result in most actin molecules being Nt-acetylated, whereas a minority is Nt-arginylated (discussed later in this review). Actins are first synthesized as precursor molecules which are further N terminally processed by successive actions of N-terminal acetyltransferases and aminopeptidases. This process was first described by Redman and Rubenstein in the early 1980s [23Redman K. Rubenstein P.A. NH2-terminal processing of Dictyostelium discoideum actin in vitro.J. Biol. Chem. 1981; 256: 13226-13229Abstract Full Text PDF PubMed Google Scholar], and only recently more details on the players have become available. The six expressed mammalian actin isoforms are divided into two categories based primarily on the nature of their unprocessed N-terminal sequences (Figure 2A) [4Vandekerckhove J. Weber K. At least six different actins are expressed in a higher mammal: analysis based on the amino-acid sequence of the amino-terminal tryptic peptide.J. Mol. Biol. 1978; 126: 783-802Crossref PubMed Scopus (532) Google Scholar]. For class I actins (nonmuscle β- and γ-actin) the initiator methionine (Met1) is directly followed by three acidic amino acids (MDDD-/MEEE-). The actin maturation process begins when the nascent N terminus is cotranslationally Nt-acetylated by NatB, which also acetylates other eukaryotic proteins beginning with MD-/ME- [26Van Damme P. et al.N-terminal acetylome analyses and functional insights of the N-terminal acetyltransferase NatB.Proc. Natl. Acad. Sci. U. S. A. 2012; 109: 12449-12454Crossref PubMed Scopus (128) Google Scholar]. Normally, acetylation of acidic N termini ensures that Met1 is retained, but in an unusual twist from nature's side the Nt-acetylated Met1 is removed by a still unidentified aminopeptidase. The neo-N terminus (DDD-/EEE-) is then Nt-acetylated by the recently identified NAA80/NatH generating the mature actin protein [27Drazic A. et al.NAA80 is actin's N-terminal acetyltransferase and regulates cytoskeleton assembly and cell motility.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: 4399-4404Crossref PubMed Scopus (102) Google Scholar, 28Wiame E. et al.NAT6 acetylates the N-terminus of different forms of actin.FEBS J. 2018; 285: 3299-3316Crossref PubMed Scopus (25) Google Scholar, 29Goris M. et al.Structural determinants and cellular environment define processed actin as the sole substrate of the N-terminal acetyltransferase NAA80.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: 4405-4410Crossref PubMed Scopus (43) Google Scholar]. For class II actins (striated and smooth muscle actins) an additional cysteine residue (MCD/E-) complicates the N-terminal processing. In this case, Met1 is cotranslationally removed by methionine aminopeptidase followed by Nt-acetylation of the exposed cysteine, presumably by NatA. Finally, an unknown aminopeptidase removes the acetylated cysteine and the processed acidic N terminus is then reacetylated, most likely by NAA80 [27Drazic A. et al.NAA80 is actin's N-terminal acetyltransferase and regulates cytoskeleton assembly and cell motility.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: 4399-4404Crossref PubMed Scopus (102) Google Scholar, 28Wiame E. et al.NAT6 acetylates the N-terminus of different forms of actin.FEBS J. 2018; 285: 3299-3316Crossref PubMed Scopus (25) Google Scholar], thereby completing the maturation process. N-terminal actin maturation gained new attention when it was discovered that the processed N terminus of β-actin (DDD-) can either be acetylated by NAA80 or arginylated by ATE1 [30Karakozova M. et al.Arginylation of β-actin regulates actin cytoskeleton and cell motility.Science. 2006; 313: 192-196Crossref PubMed Scopus (203) Google Scholar]. Nt-arginylation of β-actin is found to occur on Asp3 after the protein has undergone sequential removal of both the first and second amino acid (RDD-) (Figure 2A) [30Karakozova M. et al.Arginylation of β-actin regulates actin cytoskeleton and cell motility.Science. 2006; 313: 192-196Crossref PubMed Scopus (203) Google Scholar]. This modification profile has not been observed on any other actin isoforms. However, it would be interesting to understand why Asp3 is not further Nt-acetylated, which should be thermodynamically a more favorable reaction over the arginylation step. A recent structural analysis indicates that DD-starting actin forms a poor substrate for NAA80 [29Goris M. et al.Structural determinants and cellular environment define processed actin as the sole substrate of the N-terminal acetyltransferase NAA80.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: 4405-4410Crossref PubMed Scopus (43) Google Scholar]. Alternatively, subcellular variations in the substrate concentrations, as well as the enzyme amounts and activities, could lead to local competitions. Indeed, a recent study suggests that Nt-arginylated β-actin in mouse embryonic fibroblast (MEF) cells is concentrated at the leading edge of lamellipodia, and is thus mainly linked to active migration [31Pavlyk I. et al.Rapid and dynamic arginylation of the leading edge β-actin is required for cell migration.Traffic. 2018; 19: 263-272Crossref PubMed Scopus (21) Google Scholar]. Moreover, non-Nt-arginylated actin forms filamentous aggregates in vitro, while ATE1 KO cells show impaired lamella formation and cell migration (Figure 2B) [30Karakozova M. et al.Arginylation of β-actin regulates actin cytoskeleton and cell motility.Science. 2006; 313: 192-196Crossref PubMed Scopus (203) Google Scholar]. Acetylation enhances the negative nature of the N terminus, by neutralizing the free α-amino group, while arginylation on the other hand decreases the negative charge density. It is therefore not surprising that both modifications play a role in cytoskeleton morphology and affect actin's polymerization kinetics [27Drazic A. et al.NAA80 is actin's N-terminal acet