Post-translational modifications (PTMs) are highly dynamic and often reversible processes where a protein’s functional properties are altered by addition of a chemical group or another protein to its amino acid residues (Fig. 1). As a major cytoskeletal protein with roles in cell development, growth, motility, and intracellular trafficking, actin is a major substrate for PTMs. Actin can undergo at least 17 PTMs that include ADP-ribosylation, arginylation, dimethylation, phosphorylation, oxidation, nitration, O-GlcNAcylation, ubiquitination, SUMOylation, acetylation, carbonylation, isaspartylation, transglutamination, malonylation, glutathionylation, nitrosylation, and crosslinking/isopeptide bonding (for review, see ref. 1). Here, we will summarize what is currently known about many of these actin PTMs.
Reduction-oxidation (redox) reactions constitute a large variety of actin PTMs that include oxidation, nitrosylation, nitration, carbonylation, and glutathionylation. These PTMs occur on many different actin residues including cysteines, methionines, tyrosine, histidine, and tryptophan (Figs. 1 and 9). Actin’s five cysteine residues (Cys374, Cys272, Cys285; Cys217, Cys257) are highly vulnerable to redox modifications. The most vulnerable is Cys374 which is modified by oxidation, glutathionylation, carbonylation, and nitrosylation. PTMs of Cys374 are associated with monomer aggregation via disulfide bonds, decreased polymerization rates, increased critical concentrations, and weakening of actin filaments1. Modifications of other cysteine residues are also linked to reduced polymerization activity and altered binding of actin with actin binding proteins. Similar to cysteines, methionines are also highly vulnerable to redox PTMs and their modification reduces actin’s functionality42,43. Methionines at a variety of positions, including Met44, MET47, Met355, Met176, Met190, Met227, and Met269, are all sites of oxidation. Oxidation of Met176, Met190, and Met269 completely inhibit actin polymerization and induce rapid depolymerization of existing F-actin43.
Oxidation of actin occurs as a result of various signaling pathways throughout the cells of most, if not all, organisms ranging from bacteria to humans. One of the major pathways that mediate actin oxidation is reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) which is produced in response to extracellular signaling as well as during responses to infection or injury44,45. H2O2 inhibits actin polymerization, depolymerizes F-actin, and alters binding between actin and its binding partners44-46.
Actin can also be oxidized directly by enzymes. Mical (Cat. # MIC01), a monooxygenase, oxidizes actin in vivo and in vitro. Mical is activated upon direct binding to F-actin, oxidizing Met44 and Met47 (Cat. # MXA95) to decrease polymerization and depolymerize individual and bundled actin filaments in vivo and in vitro47,48. Active Mical also rearranges F-actin networks, making them more disorganized, creating a chaotic meshwork of short filaments47,48. It is hypothesized that Mical is able to access these “inaccessible” sites with an active site that “fits” between actin monomers48-51.
Other enzymes involved in the oxidation of actin involve the nitric oxide signaling pathway. Two redox PTMs utilizing nitric oxide signaling are nitration and S-nitrosylation. These related PTMs target tyrosine and cysteine residues, respectively, of actin52-58. Nitration (attachment of a nitrite group) on tyrosine amino acid residues leads to the functional impairment of actin and exerts consequences under both normal and pathophysiological conditions58. For example, actin in the liver and kidney cells of sickle cell disease (SCD) patients and animal models of the disease undergoes nitration53. In vivo, three tyrosine residues were nitrated, Tyr91, Tyr198, and Tyr240 as determined by mass spectrometry. In vitro, similar analyses revealed that Tyr53, Tyr198, Tyr240, and Tyr362 are nitrated, though data for Tyr362 were inconsistent53. Functionally, actin nitration reduced the critical concentration, compared to non-nitrated actin, as well as shortening the lag (nucleation) phase and accelerating filament elongation. In human and mouse kidney cells from SCD vs control populations, the altered polymerization dynamics produced F-actin staining that was disorganized and aggregated53. Actin nitration has also been implicated in endothelial barrier dysfunction, an event that can lead to vascular injury and edema54.
S-nitrosylation is a reversible PTM that is also intimately related to nitric oxide signaling and involves the covalent attachment of a nitric oxide group to a cysteine thiol56. Actin’s cysteine residues undergo S-nitrosylation under hyperoxic conditions, increasing the rate of actin polymerization. However, the result is an increased number of short actin filaments55. Hyperoxia-induced actin S-nitrosylation impairs b2 integrin clustering, an important step in responding to infection or injury55. Lu et al. reported that S-nitrosylation of actin also functions in neurotransmission, specifically in controlling neurotransmitter release56 and pain transmission in the spinal cord57. Interestingly, Lu et al.56 found that the S-nitrosylation reduced F-actin content without changing total actin levels. The authors attribute the inhibited release of neurotransmitter to the reduced F-actin content56.
Carbonylation is a form of actin oxidation that results in increased carbonyl content (e.g., aldehyde or ketone groups) of actin. This irreversible PTM leads to cross-linking and aggregation of actin as well as disruption and disassembly of F-actin networks and inhibition of polymerization59-62. Clinically, carbonylation of actin is elevated in the brains of Alzheimer’s disease patients, ischemic hearts, and cell models of inflammatory bowel disease (IBD)61. With IBD, carbonylation of actin compromises the intestinal barrier integrity which disrupts the F-actin network59. Dalle-Donne et al.61 argue that carbonylation is more than just a marker of oxidation; instead indicating severe physiological impairments related to F-actin disruption and disassembly.
Glutathionylation is another redox PTM that targets two of actin’s cysteine amino acid residues (Cys217, Cys374). Glutathionylation is a reversible PTM whereby glutathione is attached to an actin’s cysteine residue via a disulfide bond, creating glutathione disulfide. Actin glutathionylation serves to protect actin, and thereby cells, from oxidative stress63-65 (Fig. 3). For example, actin glutathionylation is believed to participate in stabilization of axons and dendrites as well as neuron survival during periods of oxidative stress64. Furthermore, actin glutathionylation influences how cells’ actin networks respond to growth factors, mediating actin polymerization and subsequent trafficking and re-arrangement of F-actin66. During oxidative stress, glutathionylation increases which decreases actin polymerization, resulting in reduced F-actin levels1,66 (Fig. 1). Besides inhibiting F-actin formation, increased glutathionylation has also been linked to abnormal rearrangement of actin filaments65,67. Upon reversal of glutathionylation, actin polymerization increases66.
It has also been suggested that actin can act as an antioxidant/scavenger to protect the cell and other proteins from oxidative stressors and oxidative metabolism61,68,69.
Figure Legend 9: Structural model of actin with individual amino acid residues highlighted in yellow that are targeted for redox-based post-translational modifications. Arrow indicates N-terminus. Structure is based on monomeric actin in its ATP-bound state, PDB identifier: 2HF4.
Arginylation is mediated by arginyltransferase (Ate1) and involves the addition of arginine on the N-terminus of beta-actin by a peptide bond2,3 (Figs. 1 & 2). Arginylation can impact actin’s function in several ways (Fig. 3). For example, arginylation increases actin polymerization3,4 and strengthens the actin filament network3, the main structural support for maintaining dendritic spine morphology and size5. Blockade/loss of arginylation is associated with defects in cell migration and myofibril contraction4,6-8 as well as collapse of leading edge lamella and reduced F-actin levels3. The leading edge collapse is specifically due to decreased N-terminally arginylated beta actin4. Besides the N-terminus, arginylation also occurs internally on the actin molecule on at least two residues1. Internal arginylation is predicted to affect polymerization and interactions between actin and actin binding proteins1. Ex vivo studies using fibroblasts cultured from Ate1 knockout mice also support a role for arginylation in actin function. Cells have slower rates of polymerization (faster nucleation/slower elongation), decreased F-actin staining, shorter actin filaments, and an increased number of intracellular actin aggregates1,3 (Fig. 1). In vivo, Ate1 knockout mice have defects in cardiovascular development9 and neural crest morphogenesis7.
Figure Legend 1: Chemical identities of actin post-translational modifications (depicted in red).
Figure Legend 2: Structural model of actin with individual amino acid residues highlighted in yellow that are targeted for the post-translational modification of arginylation. Arrow indicates N-terminus. Structure is based on monomeric actin in its ATP-bound state, PDB identifier: 2HF4.
Figure Legend 3: Post-translational modifications affect actin activity differentially. Arginylation and acetylation promote polymerization while glutathionylation decreases it. Phosphorylation can either increase or decrease actin polymerization, depending on the residue modified. Phosphorylation also affects binding between actin and actin binding proteins.
Actin is irreversibly acetylated on its N-terminus10,11 via the transfer of an acetyl group from acetyl coenzyme A to the termini of alpha-amino groups11 (Figs. 1 & 4). N-terminal acetylation is true for most types of actins12,13. In acetylation, aminopeptidases first remove the N-terminal Met (and sometimes the second amino acid) and then N-terminal acetyltransferases (NATs) modify one or more actin residues. While nonacetylated actin polymerizes and depolymerizes normally12, N-terminal acetylation is required for normal actin structure and complete functionality14,15 (Fig. 3). Specifically, N-terminal acetylation is involved with strengthening actomyosin interactions12-15 and may also influence actin ubiquitination and subsequent processing by the ubiquitin-dependent degradative molecules (for review, see refs 1 & 11).
The N-terminus of actin is not the only site for actin acetylation. At lysine residues away from the N-terminus, histone acetyltransferases (HATs; e.g., PCAF, HDAC6) interact with actin to influence actin binding and function, including transcriptional regulation16.
Figure Legend 4: Structural model of actin with individual amino acid residues highlighted in yellow that are targeted for the post-translational modification of acetylation. Arrow indicates N-terminus. Structure is based on monomeric actin in its ATP-bound state, PDB identifier: 2HF4.
ADP-ribosylation involves the transfer of ADP-ribose from nicotinamide adenine dinucleotide (NAD) to various G-actin residues1,17 (Figs. 1 and 5). ADP-ribosylation is carried out by multiple bacterial toxins and enzymes as well as mammalian enzymes. ADP-ribosylation of G-actin on Arg177 is among the most studied residues. This PTM can be mediated by many different ADP-ribosylating bacterial toxins (e.g., Clostridium botulinum C2)17. ADP-ribosylation at this residue induces cell rounding as well as inhibition of actin polymerization via steric hindrance17. Besides being unable to polymerize, ADP-ribosylated G-actin prevents further polymerization of actin filaments by capping the barbed end of the growing actin filament. ADP-ribosylation at Arg177 also impairs the binding of ATP to actin, actin’s ATPase activity, and nucleation of actin by gelsolin1,17. However, ADP-ribosylation is not strictly a negative regulator of actin dynamics and function17. The bacterial ADP-ribosyltransferase TccC3 produced by Photorhabdus luminescens targets the Thr148 actin residue, leading to increased actin polymerization, possibly due to reduced thymosin-b4 binding17.
An ADP-ribosylactin hydrolase that acts upon ADP-ribosylated actin has been described in mammalian brain, indicating that this PTM is reversible in mammals18. Upon reversal of ADP-ribosylation, F-actin was formed from the actin monomers18.
Figure Legend 5: Structural model of actin with individual amino acid residues highlighted in yellow that are targeted for the post-translational modification of ADP ribosylation. Arrow indicates N-terminus. Structure is based on monomeric actin in its ATP-bound state, PDB identifier: 2HF4.
Actin monomers can be modified by cross-linking enzymes or chemical reagents on either an intra- or intermolecular level (between monomers of the same filament vs between monomers of adjacent filaments, respectively). Among multiple amino acid residues involved in cross-linking1, some well-studied residues include Gln41 to Lys11319, Gln41 to Cys37420, and Cys374 to Lys19121 (Figs. 1 and 6). These cross-linkings are reported to decrease the speed and force generation of actomyosin without altering binding between actin and myosin or myosin’s ATPase activity22. However, a few studies have reported that actin cross-linking disrupts actomyosin binding and activation of myosin’s ATPase activity23,24. The proteins implicated in cross-linking are bacterial toxins MARTX and VgrG-117 as well as bacterial and eukaryotic transglutaminases1 (TGases). The bacterial toxins induce cross-linking of actin monomers to form polymerization-deficient dimers, trimers, and oligomers17, leading to depolymerization, fragmentation, and proteolytic degradation of F-actin. TGases are enzymes that catalyze an interaction between a glutamine acceptor residue and an amine donor to cross-link actin monomers by themselves, between filaments, or within one filament. TGases can also create actin cross-linkages using polyamine bridges. Although TGase-mediated cross-linking inhibits myosin motor activity (see above), others have reported that TGase-mediated cross-linking has positive effects. For instance, F-actin is stabilized by cross-linking25,26, as is beta-actin in the neuronal actin cytoskeleton27.
Figure Legend 6: Structural model of actin with individual amino acid residues highlighted in yellow that are targeted for the post-translational modification of cross-linking. Arrow indicates N-terminus. Structure is based on monomeric actin in its ATP-bound state, PDB identifier: 2HF4.
Modifications by attachment of polysaccharide chains or glycans to proteins (glycosylation) can occur via multiple mechanisms, including N-linked and O-linked glycosylation, glycosylation with O-linked beta-N-acetylglucosamine (O-GlcNAcylation), and glycation. Typical glucose modifications of actin are by O-GlcNAcylation and glycation as N- and O-linked glycosylation are PTMs characteristic of lumenal proteins and proteins involved in the secretion and trafficking of proteins along membrane-associated pathways1. That is not to say that actin cannot be glycosylated as in utero exposure of rats to alcohol results in glycosylated actin28.
Nuclear and cytoplasmic actin is also modified by O-GlcNAcylation29, a reversible PTM believed to modulate actin’s role in cardiac and skeletal muscle contraction, cardiac health, and diabetes1,30,31 (Figs. 1 and 7). Attachment of O-linked beta-N-acetylglucosamine (O-GlcNAc) onto serine or threonine residues of nuclear and cytoplasmic proteins is controlled by two highly conserved enzymes, O-GlcNAc transferase (OGT) and b-N-acetylglucosaminidase (O-GlcNAcase)32. O-GlcNAc cycles at rates similar to that of O-phosphate in response to various cellular stimuli29,32.
Glycation, or non-enzymatic glycosylation, is another glucose-based modification that results from the non-enzymatic attachment of glucose to lysine residues. Glycation is a PTM linked to diabetes as actin in diabetic patients and animal models of diabetes is susceptible to this PTM33-36. Similar effects of glucose exposure have been reported in cell models of diabetes with decreased activation of myosin ATPase, reduced polymerization, and inhibition of DNAse I36-38.
Figure Legend 7: Structural model of actin with individual amino acid residues highlighted in yellow that are targeted for glucose-based post-translational modifications. Arrow indicates N-terminus. Structure is based on monomeric actin in its ATP-bound state, PDB identifier: 2HF4.
Methylation of actin is an irreversible PTM that occurs on multiple residues, including His7339, which has a part in regulating actin’s interdomain flexibility and stability through stabilization of F-actin (for review, see ref. 1) (Figs. 1 and 8). Another actin residue that undergoes methylation is Lys326 as well as specific actin residues that have been arginylated40. Methylation of arginylated actin residues (a double PTM on the same residue) is likely involved in regulating chromatin structure, gene expression, and nuclear proteins40. Such methylation could also protect arginylated residues from further modification40. Actin methylating enzymes (i.e., protein methylases) have been described39,41, though the exact amino acid residues they target are unknown.
Figure Legend 8: Structural model of actin with individual amino acid residues highlighted in yellow that are targeted for the post-translational modification of methylation. Arrow indicates N-terminus. Structure is based on monomeric actin in its ATP-bound state, PDB identifier: 2HF4.
Actin has at least 35 amino acid residues that can be modified by phosphorylation and this PTM can exert both negative and positive effects on polymerization (Figs. 1, 3, & 10). For example, actin’s Tyr53 residue in the slime mold Dictyostelium is phosphorylated, which interferes with polymerization, likely through a disruption of actin subunit-subunit contact70,71. Conversely, in the slime mold Physarum, actin fragmin kinase (AFK), a calcium-dependent enzyme, phosphorylates Thr201-203, leading to elongation of the actin filaments72-75. This elongation is believed to be a result of reduced interactions between fragmin and actin. Fragmin is related to the severing protein gelsolin and as such, controls filament length72-75. The effect of Thr phosphorylation is reversed by protein phosphatases PP1 and PP2A76. In both organisms, the changes in actin phosphorylation states are associated with cytoskeletal responses to extracellular events (e.g., locomotion, phagocytosis, signal transduction) and transition into a state of dormancy1,70-73.
In mammals, proteomic analyses have revealed that multiple kinases phosphorylate actin and vary by cell type, disease conditions, and external stimuli. Unfortunately, many of the studies are correlational and do not report a direct relationship between a given kinase and actin phosphorylation1. For example, Ser and Tyr residues on actin are phosphorylated in response to insulin via unknown kinases, leading to reduced DNAse I binding1 (Fig. 1). Likewise, activation of the p21-activated kinase PAK1 leads to actin phosphorylation which is correlated with loss of stress fibers and altered F-actin localization77. Similarly, Src kinase-driven phosphorylation of actin impairs actin polymerization1,78. Several known actin kinases are casein kinase I1,79, cAMP-dependent protein kinase (PKA), and calcium/phosphoinositide-dependent protein kinase (PKC)80,81. Casein kinase I phosphorylates actin similar to AFK (targets Thr and Ser residues and is calcium-dependent). PKA and PKC act in an opposing manner with the former impairing polymerization and the latter stimulating it82,83 (Fig. 1).
Figure Legend 10: Structural model of actin with individual amino acid residues highlighted in yellow that are targeted for the post-translational modification of phosphorylation. Arrow indicates N-terminus. Structure is based on monomeric actin in its ATP-bound state, PDB identifier: 2HF4.
Ubiquitination of proteins involves either the addition of one (mono) or more (poly) ubiquitin/ubiquitin-like proteins (Figs. 1 & 11). Actin undergoes both modifications and monoubiquitination is believed to direct subcellular actin localization and confer stability84,85 while polyubiquitination signals actin proteins bound for degradation. Actin is mono- and/or polyubiquitinated by the ubiquitin ligases MuRF1, UbcH5, and Trim3286-89, leading to predictable decreases in actin89. The degradation of ubiquitinated actin is correlated with muscle remodeling and atrophy87-89. The actin binding protein myosin may protect ubiquitinated actin from degradation88. Ubiquitination also serves to degrade actin proteins that have been incorrectly modified by other PTMs such as arginylation90. Besides ubiquitination, actin is also targeted by small ubiquitin-like modifiers (SUMO), which are involved in the nuclear localization of actin91-93 (Figs. 1 and 12). The SUMO proteins are small (approximately 11 kDa) and the modification is covalent and reversible. There are multiple SUMO isoforms and Hofmann et al.93 reported that both SUMO2 and SUMO3 modify nuclear actin through an interaction involving amino acids Lys68 and Lys284.
Figure Legend 11: Structural model of actin with individual amino acid residues highlighted in yellow that are targeted for the post-translational modification of ubiquitination. Arrow indicates N-terminus. Structure is based on monomeric actin in its ATP-bound state, PDB identifier: 2HF4.
Figure Legend 12: Structural model of actin with individual amino acid residues highlighted in yellow that are targeted for the post-translational modification of SUMOylation. Arrow indicates N-terminus. Structure is based on monomeric actin in its ATP-bound state, PDB identifier: 2HF4.
Actin is a major cytoskeletal protein whose function is modulated by a variety of PTMs (Figs. 1-12). Despite actin’s relevance in all aspects of cell biology, our current understanding of how at least 17 different PTMs affect actin polymerization, stability, and binding is not complete. As new PTM tools are developed, we can look forward to greatly advancing our understanding of actin PTMs.
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