Myosin IIa signal von Willebrand factor release

Abstract

In this issue of Blood, Li et al demonstrate that phosphorylation of distinct serine residues on the motor protein myosin IIa regulates the peripheral distribution of Weibel-Palade bodies and the cyclic adenosine monophosphate (cAMP)–induced secretion of von Willebrand factor.¹

Endothelial cells (ECs) of the vascular bed provide a regulated barrier that controls physiological processes such as hemostasis, thrombosis, and inflammation. Intrinsic to these functional processes is the regulated secretion of hemostatic and inflammatory modulators, including von Willebrand factor (VWF) from the endothelial Weibel-Palade bodies (WPBs). Matured WPB tagged with a small GTPase Rab27a tend to localize near the periphery of the EC, and the immature WPB accumulate near the nucleus.² Regulated secretion of VWF involves the translocation of WPB from the perinuclear region in the cytoplasm toward the cell membrane and the subsequent fusion of WPB with the plasma membrane. The importance of WPB localization is underscored by the observation that peripheral but not perinuclear distributed WPBs secrete VWF from the ECs in response to secretagogues that activate cAMP signaling,³ yet the mechanisms that govern the peripheral localization of WPB in EC are not fully understood.

One mechanism identified in Li et al’s article involves myosin IIa, a protein that belongs to a class of cytoskeletal molecular motors and is expressed exclusively in nonmuscle cells. Myosin II motor complex can transduce free energy into mechanical work and consist of myosin II heavy chain homodimer, 2 essential light chains and 2 regulated light chains.⁴ Myosin heavy chain has 3 isoforms, namely, myosin IIa, myosin IIb, and myosin IIc, which are expressed in a tissue- specific manner by MYH9, MYH10, and MYH14 genes, respectively. The concept that myosin II can modulate VWF secretion is not entirely new. Nightingale et al proposed that after the fusion of WPB with cell membrane, a dynamic ring of actin and myosin II appears around the fused WPB. The actomyosin contractility triggered by myosin II activity squeezes VWF out of the membrane-fused WPB.⁵ This observation suggests a role for myosin II in VWF secretion after the fusion of WPB. However, the contribution of myosin IIa isoform in VWF secretion remains unknown.

In the article by Li et al, the investigators reveal that myosin IIa promotes the distribution of peripheral WPB and facilitates VWF secretion. In vitro and in vivo release of VWF induced by epinephrine was decreased in myosin IIa–depleted human umbilical vein endothelial cells and in an inducible endothelial- specific myosin IIa null mouse model. Epinephrine-challenged myosin IIa null mice demonstrated prolonged hemostasis and impaired thrombosis induced by a FeCl₃ injury. Mechanistically, depletion of myosin IIa, but not myosin IIb, isoform and inhibition of myosin II activity diminish the peripheral localization of Rab27a-positive WPB along focal adhesion–anchored stress fibers. Importantly, the authors demonstrate that in response to cAMP signaling, myosin II activity is critical for the formation of a functional actin framework around WPB before the fusion of WPB with the cell membrane. Furthermore, in a biochemical approach, the authors detect an interaction of myosin IIa with a focal adhesion protein zyrin and show that myosin IIa–zyrin protein interaction is important for cAMP-induced VWF secretion. Lastly, using phosphomimetic and phosphoablatant mutants of myosin IIa, the authors identify phosphorylation of myosin IIa at serine 1943 as essential for maintaining the peripheral localization of WPB and that phosphorylation of serine 1916 by casein kinase 2 is required for VWF secretion in a zyrin-dependent fashion.

This detailed and interesting study provides original insights into the molecular machineries that govern WPB exocytosis. First, the authors identify a new myosin IIa interacting protein, zyrin, and establish the functional importance of this protein complex in VWF secretion. Second, they demonstrate that specific serine phosphorylation on myosin IIa is essential for cAMP-induced VWF secretion. This phosphorylation is on the tail of the myosin heavy chain and is distinct from the extensively studied threonine 18/serine19 phosphorylation of the myosin light chain, which noncovalently associates in the myosin II complex. Previous studies have examined the role of other motor proteins in WPB localization and VWF secretion. For example, Roja et al showed that myosin Va prevents the secretion of less matured VWF-containing WPB.⁶ Cytoskeletal motor dynein–dynactin complex facilitates WPB clustering on a microtubule network around the perinuclear region in response to cAMP signaling and presumably limits VWF release.⁷ Thus, these studies performed under disparate experimental conditions suggest that motor proteins (myosin IIa, Va, and dynein) may associate with either mature or immature pools of WPB and positively or negatively regulate VWF secretion.

The observation that loss of myosin IIa but not myosin IIb disrupts the peripheral localization of WPB is intriguing. These data raise additional questions. How does myosin IIa regulate positioning of WPB? Do myosin IIa and IIb in EC have discrete functions? Could the myosin isoforms interact with specific effector proteins to perform explicit functions? There is evidence that MyRIP (myosin- and Rab27a-interacting protein) is recruited to Rab27a-bound WPB and that depletion of MyRIP decreased peripheral localization of WPB,² but it is unknown whether MyRIP interacts with myosin IIa, IIb, or both.

The interest in myosin IIa is not limited to academics, because mutations in the MYH9 gene (myosin IIa) are linked to a number of autosomal dominant disorders, collectively called MYH-9–related disease (MYH9RD) (May-Hegglin anomaly and Sebastian, Epstein, and Fechtnes syndromes). This is described as a platelet disorder, with patients exhibiting congenital macrothrombocytopenia and mild bleeding phenotype.⁸ In this context, the data from the authors beg the question, is endothelial VWF secretion impaired in these patients, and if yes, does it contribute to the mild bleeding phenotype? Lastly, it remains to be determined whether the mechanisms identified by the authors are also applicable in another VWF-secreting cell type: platelets.