Extracellular Phosphorylation of Collagen XVII by Ecto-Casein Kinase 2 Inhibits Ectodomain Shedding

Abstract

Ecto-phosphorylation is emerging as an important mechanism to regulate cellular ligand interactions and signal transduction. Here we show that extracellular phosphorylation of the cell surface receptor collagen XVII regulates shedding of its ectodomain. Collagen XVII, a member of the novel family of collagenous transmembrane proteins and component of the hemidesmosomes, mediates adhesion of the epidermis to the dermis in the skin. The ectodomain is constitutively shed from the cell surface by metalloproteinases of the ADAM (a disintegrin and metalloproteinase) family, mainly by tumor necrosis factor-␣ converting enzyme (TACE). We used biochemical, mutagenesis, and structural modeling approaches to delineate mechanisms controlling ectodomain cleavage. A standard assay for extracellular phosphorylation, incubation of intact keratinocytes with cell-impermeable [␥-32 P]ATP, led to collagen XVII labeling. This was significantly diminished by both broad-spectrum extracellular kinase inhibitor K252b and a specific casein kinase 2 (CK2) inhibitor. Collagen XVII peptides containing a putative CK2 recognition site were phosphorylated by CK2 in vitro, disclosing Ser 542 and Ser 544 in the ectodomain as phosphate group acceptors. Phosphorylation of Ser 544 in vivo and in vitro was confirmed by immunoblotting of epidermis and HaCaT keratinocyte extracts with phosphoepitopespecific antibodies. Functionally, inhibition of CK2 kinase activity or mutation of the phosphorylation acceptor Ser 544 to Ala significantly increased ectodomain shedding, whereas overexpression of CK2␣ inhibited cleavage of collagen XVII. Structural modeling suggested that the phosphorylation of serine residues prevents binding of TACE to its substrate. Thus, extracellular phosphorylation of collagen XVII by ecto-CK2 inhibits its shedding by TACE and represents novel mechanism to regulate adhesion and motility of epithelial cells.Cells need a versatile, fast mechanism to respond to changes in their microenvironment during development, growth, and regeneration. Regulated proteolysis on the cell surface offers a post-translational mechanism to remove or structurally modify surface associated proteins at any time (1). For a multitude of type I and type II transmembrane proteins, such as receptors, cell adhesion molecules, and growth factors, soluble forms have been identified that have functions distinct from their membrane-bound counterparts. These are often generated through ectodomain shedding, a general mechanism that influences interactions of different cell types with the environment in a broad spectrum of biological and pathological processes (1, 2). However, regulation of the ectodomain shedding remains poorly understood, namely, targeting of the sheddase to particular substrate depending on biological context.One prime example in this context is collagen XVII, a prototype of the novel family of collagenous transmembrane proteins (3). It is a structural component of hemidesmosomes that mediate adhesion of the epidermal keratinocytes to the underlying...

Ectodomain Shedding Generates Neoepitopes on Collagen XVII, the Major Autoantigen for Bullous Pemphigoid

“…Production of Abs to candidate neoepitopes on the shed ectodomain of collagen XVII Based on previously described candidate cleavage sites on collagen XVII (19)(20)(21), four different short peptides, 7-9 aa in length, and with nonblocked N termini, were prepared (Fig. 1A).…”

“…One study used protein extracted from normal human skin and suggested N-terminal cleavage at Ala 531 (19 in immortalized HaCat keratinocyte and DJM-1 cell lines (20). In addition, structural modeling of ADAM17 with phosphorylated collagen XVII predicted Leu 540 to be a cleavage site (21). In contrast, a further study showed that a mAb with an epitope within the amino acid stretch of Ser 515 to Arg 523 recognized the shed ectodomain of collagen XVII (22), indicating that ectodomain shedding of collagen XVII could occur at different position(s), probably in a context-dependent manner.…”

“…In concert, Ab 83457, which was produced by immunization against the peptide Leu . Ectodomain shedding of collagen XVII is catalyzed by ADAM proteases, especially by ADAM17 (15,17,21), 9, and 10 (15, 18). ADAMs do not require a specific cleavage consensus sequence, but cleave their transmembrane substrates at a defined length from the cell surface (17,31).…”

Ecto-Phosphorylation and Regeneration of the Adult Central Nervous System

“…Furthermore, some of extracellular matrix proteins are known to be phosphorylated [26,[30][31][32]. Yalak and Vogel identified 770 different phosphorylation sites in 66 extracellular proteins or in proteins with extracellular domain by annotation of secreted phosphorylated protein data available in public repositories [33].…”

“…The serine 544 in the extracellular domain of collagen XVII is phosphorylated by CK2 and the phosphorylation inhibits shedding of the extracellular domain by metalloproteases of the A disintegrin and metalloproteinase (ADAM) family [32]. Although the exact function of collagen XVII is unknown, collagen XVII is expressed in the CNS and its distribution is changed in neurodegenerative disorders [63].…”