Post-translational arginylation as a novel regulator of platelet function

In this issue of Haematologica, Lian et al. show that megakaryocyte-specific loss of arginyltransferase (ATE1)-mediated post-translational arginylation leads to enhanced clot retraction and in vivo thrombus formation in mice, due to enhanced myosin regulatory light chain (RLC) phosphorylation in platelets.1

Platelets are small, discoid-shaped cells circulating in the bloodstream. After vascular injury, platelets are recruited to the exposed subendothelial extracellular matrix that triggers platelet activation to seal wound sites by the formation of a hemostatic plug. Consequently, excessive bleeding is prevented under normal circumstances. However, if thrombus formation is uncontrolled, it may lead to vessel occlusion and to life-threatening events, such as myocardial infarction and stroke.2

Platelet activation involves a large number of platelet surface receptors, signaling molecules, and cytoskeletal-modifying proteins, as well as their complex interactions. Platelet signaling requires a cascade of intracellular protein post-translational modifications, of which phosphorylation is probably the best studied. Mass spectrometry analysis revealed that more than 270 proteins are phosphorylated in human platelets.3 The number of studies on how post-translational modifications influence platelet biology is increasing, demonstrating that these modifications constitute an emerging, biologically significant field.

Post-translational arginylation, or tRNA-dependent addition of the amino acid arginine to proteins, was discovered more than 40 years ago. However, it remained a comparatively less-known post-translational modification. Arginylation was once thought to play a singular role in the N-end rule pathway of protein degradation. However, recent evidence shows that protein arginylation also regulates a wide variety of crucial biological processes, including embryogenesis, cardiovascular development, angiogenesis, and neural crest cell migration, which are just beginning to be understood (reviewed by Saha et al.4). For example, arginylation of β-actin has been found to regulate lamellipodial formation at the leading edge in fibroblasts, suggesting that similar functions of β-actin in other cell types may also require arginylation. Thus, arginylation is emerging as a regulator protein of function that is reminiscent of phosphorylation.4,5

Protein arginylation is mediated by the arginyltransferase ATE1 (for Arginine Transfer Enzyme 1), an enzyme present in all eukaryotic cells.4 Arginylation requires no additional factors besides the ATE1 enzyme, the charged arginine-tRNA, and the protein substrate. ATE1 can transfer arginine not only to the N-terminus, but also to internal sites in a protein substrate, and is capable of self-arginylation, which is likely involved in its regulation.6 Every organism, from yeasts to humans, contains the ATE1 gene, which encodes a single protein in lower eukaryotes, and multiple isoforms in higher species. The mouse Ate1 gene encodes four ATE1 isoforms, i.e. ATE1-1/4, produced by different combinations of four alternatively spliced exons 1 and 2 and exons 8 and 9. ATE1-1/2 have higher activity and more substrate specificity than ATE1-3/4. ATE1-2 is the most ubiquitousty expressed ATE1 isoform in different mouse tissues.7 The essential physiological significance of protein arginylation in vivo was shown by the generation of Ate1-null mice, which results in embryonic lethality with defects in cardiovascular development and angiogenesis.8,9

Lian et al. demonstrate that the ubiquitous ATE1-2 is the main isoform expressed in mouse platelets.1 The total amount of ATE1 protein in platelets is similar to that in fibroblasts. The authors further capitalized on megakaryocyte-specific Ate1-null mice to investigate the role of ATE1-mediated protein arginylation in platelet biology, which had been completely unexplored before. Ate1^fl/fl Pf4-Cre mice do not suffer from spontaneous bleeding. Consistently, lack of ATE1 function is dispensable for platelet biogenesis, secretion, aggregation and spreading. In contrast to the situation in fibroblasts, ATE1 is not essential for β-actin assembly in platelets, suggesting that ATE1 might have other targets in platelets. Consequently, Lian et al. tested platelets for other candidate proteins as possible targets for arginylation and describe more than 20 proteins that are arginylated in platelets, including myosin essential light chain ELC/MYL6 on Val64 and myosin heavy chain MYH9 on Leu1844.

Myosin is a well-described protein that is involved in mediating contractile forces.10 Thus, Ate1-null platelets were analyzed for the generation of contractile forces, a highly cytoskeletal-dependent process. The authors found that Ate1-null platelets exert enhanced contractile forces, as evidenced by accelerated clot retraction, and were able to pinpoint the molecular mechanism by showing that phosphorylation of myosin RLC on Ser19, but not on Thr18 is enhanced in thrombin-stimulated Ate1-null platelets. Furthermore, ATE1 and myosin interact in mouse platelets, demonstrating that ATE1 can regulate myosin function. The authors hypothesize that addition of the bulky arginine on ELC/MYL6 Val64 might impair the access of myosin light chain kinase to myosin RLC Ser19 (Figure 1).

Figure 1.Hypothetical model. (A) The arginyltransferase ATE1 transfers the amino acid arginine (Arg) to myosin essential light chain (ELC) Val64 and myosin heavy chain MYH9 Leu1844. Myosin light chain kinase (MLCK) phosphorylates myosin regulatory light chain (RLC) Ser19 and activates it. (B) Myosin RLC Ser19 phosphorylation is increased in Ate1-null platelets, as access of MLCK to its target is influenced by the lack of the bulky arginine addition to myosin ELC Val64. This in turn enhances myosin activity, leading to increased clot retraction, faster thrombus formation and shorter bleeding time in megakaryocyte-specific Ate1-null mice.

It was previously reported that the contractile mechanisms in platelets are critical for maintaining the integrity of a hemostatic plug at wound sites independently of thrombin and fibrin generation and that the tight packing of platelets was reversed after blocking MYH9 activity by using the myosin inhibitor, blebbistatin.11 In agreement with this, Ate1-null platelets with enhanced contractile behavior formed thrombi after FeCl₃-induced carotid artery injury and arrested bleeding faster than control platelets. These data support previous observations that myosin-mediated contractile forces contribute to proper thrombus formation and may have important, clinically related implications for patients with MYH9-related platelet disorders suffering from altered hemostatic function.12

In conclusion, Lian et al. convincingly revealed a so far unknown role for arginylation, a lesser known post-translational modification, in platelet biology and demonstrate that there is a hierarchical network of post-translational modifications, with myosin ELC/MYL6 arginylation regulating the level of myosin RLC phosphorylation. Whether this is a general regulatory mechanism in cells remains to be determined. For example, the authors identified the arginylation of the cytoskeletal and scaffold protein filamin A in platelets. Filamin A plays a critical role in platelet morphology and signaling, as it cross-links actin filaments, tethers the von Willebrand factor receptor glycoprotein Ib-IX-V complex and integrins to the underlying actin cytoskeleton, and serves as a scaffold for signaling intermediates, e.g. the tyrosine kinase Syk13 (reviewed by Falet et al.14). Filamin A arginylation occurs on three different sites, i.e. Pro2151, Phe2311 and Tyr2501, among which Pro2151 is particularly important, as it is located near Ser2152, a major phosphorylation site and a possible regulator of integrin binding. It will be interesting to investigate if and how ATE1-mediated arginylation influences the function of the more than 20 arginylated proteins, for example filamin A, in platelets. Thus, we are just at the beginning of understanding the importance of arginylation in platelet biology.

Acknowledgments

This work was supported by Deutsche Forschungsgemeinschaft (DFG) postdoctoral fellowship BE 5084/1-1 (to MB) and National Institutes of Health (NIH) grant HL059561 (to HF).

Footnotes

• Markus Bender, PhD, is a Postdoctoral Fellow at Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA. Hervé Falet, PhD, is Instructor in Medicine at Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA. His main field of interest is platelet and megakaryocyte biology.
• Financial and other disclosures provided by the author using the ICMJE (www.icmje.org) Uniform Format for Disclosure of Competing Interests are available with the full text of this paper at www.haematologica.org.