In vitro phosphorylation of von Willebrand factor by FAM20c enhances its ability to support platelet adhesion

Da, Qi; Han, Hui; Valladolid, Christian; Fernández, María; Khatlani, Tanvir; Pradhan, Subhashree; Nolasco, Jennifer; Matsunami, Risë K.; Engler, David; Crúz, Miguel A.; Vijayan, K. Vinod

doi:10.1111/jth.14426

Cited by 11 publications

(13 citation statements)

References 30 publications

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“…Fam20C has been found to directly phosphorylate fibrinogen alpha and gamma chains in vitro ( 56 ), and further work is needed to define the physiological roles of the phosphorylation events. On a similar note, Fam20C phosphorylates the A2 domain of von Willebrand factor (vWF) on two SxE sites, pSer1517 and pSer1613 ( 90 ). The modifications promote platelet adhesion to sites of vascular injury and helps in coagulation ( 90 ).…”

Section: Fam20c the Secreted Golgi Casein Kinasementioning

confidence: 96%

The ABCs of the atypical Fam20 secretory pathway kinases

Worby

Mayfield

Pollak

et al. 2021

Journal of Biological Chemistry

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The study of extracellular phosphorylation was initiated in late 19th century when the secreted milk protein, casein, and egg-yolk protein, phosvitin, were shown to be phosphorylated. However, it took more than a century to identify Fam20C, which phosphorylates both casein and phosvitin under physiological conditions. This kinase, along with its family members Fam20A and Fam20B, defined a new family with altered amino acid sequences highly atypical from the canonical 540 kinases comprising the kinome. Fam20B is a glycan kinase that phosphorylates xylose residues and triggers peptidoglycan biosynthesis, a role conserved from sponges to human. The protein kinase, Fam20C, conserved from nematodes to humans, phosphorylates well over 100 substrates in the secretory pathway with overall functions postulated to encompass endoplasmic reticulum homeostasis, nutrition, cardiac function, coagulation, and biomineralization. The preferred phosphorylation motif of Fam20C is SxE/pS, and structural studies revealed that related member Fam20A allosterically activates Fam20C by forming a heterodimeric/tetrameric complex. Fam20A, a pseudokinase, is observed only in vertebrates. Loss-of-function genetic alterations in the Fam20 family lead to human diseases such as amelogenesis imperfecta, nephrocalcinosis, lethal and nonlethal forms of Raine syndrome with major skeletal defects, and altered phosphate homeostasis. Together, these three members of the Fam20 family modulate a diverse network of secretory pathway components playing crucial roles in health and disease. The overarching theme of this review is to highlight the progress that has been made in the emerging field of extracellular phosphorylation and the key roles secretory pathway kinases play in an ever-expanding number of cellular processes.

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Section: Fam20c the Secreted Golgi Casein Kinasementioning

confidence: 96%

The ABCs of the atypical Fam20 secretory pathway kinases

Worby

Mayfield

Pollak

et al. 2021

Journal of Biological Chemistry

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“…Furthermore, FAM20C-mediated phosphorylation of oocyte secreted bone morphogenetic protein 15 and growth differentiation factor 9 is essential for regulation of their activity in folliculogenesis and ovulation [ 6 ]. Likewise, FAM20C-mediated phosphorylation of von Willebrand factor increased platelet adhesion [ 7 ], and lack of FAM20C phosphorylation of histidine-rich calcium binding protein causes ventricular arrhythmia [ 8 ].…”

Section: Introductionmentioning

confidence: 99%

Ras-transformation reduce FAM20C expression and osteopontin phosphorylation

et al. 2020

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Family with sequence similarity 20, member C (FAM20C) is the main kinase of secreted phosphoproteins, including the multifunctional protein and cytokine, osteopontin (OPN). The phosphorylation of OPN varies greatly among cell types, tissues and species, and the different phospho-isoforms contribute to the multifunctionality of the protein. Expression of OPN is increased in human malignancies, and less phosphorylated isoforms of the protein have been associated with this phenotype. Here, we compared OPN from ras-transformed fibroblasts with that from their non-transformed parental cells, and found that OPN was less phosphorylated after ras-transformation. Furthermore, we demonstrated that expression of FAM20C mRNA was reduced five-fold in ras-transformed fibroblasts compared to non-transformed fibroblasts. Transfection with FAM20C of the ras-transformed fibroblasts restored the FAM20C mRNA expression but the phosphorylation of OPN was not increased proportionally. Likewise, the mRNA level of FAM20C was reduced in the malignant ras-transformed mammary cell line MCF10ACA1a compared to its non-transformed parental cell line MCF10A. These results suggest that expression of the FAM20C kinase is reduced after oncogenic ras-transformation, which potentially affects the phosphorylation of secreted phosphoproteins.

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“…The A domains have three S‐x‐E sites that were surveyed in the study. They found that both S1517 and S1613 in the A2 domain were phosphorylated by active FAM20c in vitro . When assessing the phosphorylation state of VWF purified from healthy donor plasma, they validated that S1613 could be phosphorylated in vivo, though the relative abundance compared to that of nonphosphorylated S1613 is not clear.…”

mentioning

confidence: 99%

“…A recent investigation performed by Da et al provides evidence that von Willebrand factor (VWF) can be phosphorylated in the A2 domain, a modification that has never before been observed . VWF is a multimeric glycoprotein that mediates platelet adhesion to the endothelium upon vascular injury, a process critical for hemostasis.…”

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confidence: 99%

“…First, how does phosphorylation of S1613 affect the activity of VWF? Da et al reported no observable changes in ADAMTS13‐mediated proteolysis between VWF molecules bearing phosphorylated or nonphosphorylated S1613, which suggests that phosphorylation of S1613 would not impact hemostatic potential or thrombotic risk based on its length. A key observation in the study is that VWF fragments with phosphorylated S1613 support enhanced platelet adhesion in the presence of shear.…”

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confidence: 99%

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FAM‐tastic phospho‐regulation of von Willebrand factor activity

Legan

2019

Journal of Thrombosis and Haemostasis

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A recent investigation performed by Da et al provides evidence that von Willebrand factor (VWF) can be phosphorylated in the A2 domain, a modification that has never before been observed. 1 VWF is a multimeric glycoprotein that mediates platelet adhesion to the endothelium upon vascular injury, a process critical for hemostasis.Dysregulation of VWF can cause myriad bleeding and clotting diseases or complications including but not limited to von Willebrand disease; thrombotic thrombocytopenic purpura; and hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome. [2][3][4][5] VWF activity is regulated primarily by hydrodynamic shear forces in the bloodstream; however, VWF levels and activity are also influenced by other endogenous factors. For example, a disintegrinlike and metalloproteinase with thrombospondin type-1 motif member 13 (ADAMTS13) specifically targets a Tyr1605-Met1606 scissile bond within the A2 domain of VWF to generate shorter multimers, reducing the relative abundance of high-molecular-weight VWF multimers that more readily bind platelet receptor GPIbα to promote platelet adhesion. 6 Covalent modifications that regulate VWF activity and plasma levels include O-linked and N-linked glycosylation as well as oxidation. Mutagenesis of O-linked glycosylated residues on the N-terminal and C-terminal flanking regions of the VWF A1 domain leads to enhanced collagen and platelet binding. 7 N-linked glycosylation of VWF has been shown to modulate proteolysis of VWF multimers by ADAMTS13 as removal of these glycans undergoes enhanced cleavage. 8 Similarly, trimming of N-linked glycans and removal of negatively charged sialic acid (or desiaylation) on both N-linked and O-linked glycans result in accelerated clearance of VWF in mice. 9 The effects of desialylation of VWF are particularly important in the context of infection, in which neuraminidase from a pathogen desialylate and subsequently clear VWF in the bloodstream. 10 Oxidation of VWF may also pose an increased thrombotic risk. Release of neutrophil oxidants during inflammation enhances oxidation of methionine residues, particularly that of M1606 in the A2 domain, reducing ADAMTS13-mediated proteolysis of VWF multimers as evidenced through in vitro studies and studies with plasma from patients with sickle cell disease. 11 In this issue, Da et al demonstrate a potentially overlooked covalent modification of VWF by showing that the A2 domain can be phosphorylated by a secreted extracellular kinase family with sequence similarity 20, member C (FAM20c). This kinase phosphorylates proteins with an S-x-E motif and is critical for healthy bone and tooth development due to its role in tissue mineralization. 12Moreover, FAM20c was previously characterized to phosphorylate more than 100 extracellular proteins including kininogen, ADAM10, and von Willebrand factor A-domain containing protein 1, 13 lending support to the possibility that the A domains of VWF could also be phosphorylated.Da et al employed in vitro kinase studies and mass spectrometry ana...

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In vitro phosphorylation of von Willebrand factor by FAM20c enhances its ability to support platelet adhesion

Abstract: comes indicate that VWF can be phosphorylated by FAM20c in vitro, and this novel post-translational modification enhances the adhesiveness of VWF to platelets.

Cited by 11 publications

References 30 publications

The ABCs of the atypical Fam20 secretory pathway kinases

The ABCs of the atypical Fam20 secretory pathway kinases

Ras-transformation reduce FAM20C expression and osteopontin phosphorylation

FAM‐tastic phospho‐regulation of von Willebrand factor activity

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