All members of the herpesviridae contain within their large tegument protein a cysteine protease module that displays deubiquitinating activity. We report the crystal structure of the cysteine protease domain of murine cytomegalovirus M48 (M48(USP)) in a complex with a ubiquitin (Ub)-based suicide substrate. M48(USP) adopts a papain-like fold, with the active-site cysteine forming a thioether linkage to the suicide substrate. The Ub core participates in an extensive hydrophobic interaction with an exposed beta hairpin loop of M48(USP). This Ub binding mode contributes to Ub specificity and is distinct from that observed in other deubiquitinating enzymes. Both the arrangement of active-site residues and the architecture of the interface with Ub lead us to classify this domain as the founding member of a previously unknown class of deubiquitinating enzymes.

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Migration of keratinocytes requires a regulated and dynamic turnover of hemidesmosomes (HDs). We and others have previously identified three serine residues on the integrin ␤4 cytoplasmic domain that play a critical role in the regulation of HD disassembly. In this study we show that only two of these residues (Ser-1356 and Ser-1364) are phosphorylated in keratinocytes after stimulation with either PMA or EGF. Furthermore, in direct contrast to previous studies performed in vitro, we found that the PMA-and EGF-stimulated phosphorylation of ␤4 is not mediated by PKC, but by ERK1/2 and its downstream effector kinase p90RSK1/2. EGF-stimulated phosphorylation of ␤4 increased keratinocyte migration, and reduced the number of stable HDs. Furthermore, mutation of the two serines in ␤4 to phospho-mimicking aspartic acid decreased its interaction with the cytoskeletal linker protein plectin, as well as the strength of ␣6␤4-mediated adhesion to laminin-332. During mitotic cell rounding, when the overall cell-substrate area is decreased and the number of HDs is reduced, ␤4 was only phosphorylated on Ser-1356 by a distinct, yet unidentified, kinase. Collectively, these data demonstrate an important role of ␤4 phosphorylation on residues Ser-1356 and Ser-1364 in the formation and/or stability of HDs. Hemidesmosomes (HDs)3 are specialized junctional complexes that mediate firm adhesion of epithelial cells to the underlying basement membrane. Two types of HDs have been characterized: type I and II (1). Type I (classical) HDs are present in squamous and complex epithelia, such as the skin and the bladder. They contain integrin ␣6␤4, plectin, the bullous pemphigoid antigens 180 (BP180) and 230 (BP230), and the tetraspanin CD151 (2). Type II HDs lack BP180 or BP230 and are present in simple epithelia, such as the intestine (3). As the integrin ␣6␤4 binds to Ln-332 in the extracellular matrix (ECM) and associates intracellularly with plectin, which in turn interacts with the keratin filament system, a protein complex is formed that protects the cell against mechanical stress. The importance of this linkage for epidermal-dermal cohesion is substantiated by the finding that in both humans and genetically modified mice, mutations in the genes for these proteins that either prevent their expression or function, result in a skin blistering disorder known as epidermolysis bullosa (2, 4).The primary interaction between plectin and ␤4 occurs through the first pair of fibronectin type III (FnIII) domains and a small part of the connecting segment (CS) of ␤4 and the actin binding domain of plectin (plectin-ABD) (5-7). Indeed, mice carrying a specific deletion of the C-terminal portion of the ␤4 cytoplasmic domain, which still contains the plectin-ABD binding site, can still form normal HDs (8). However, binding of ␤4 to the plectin-ABD is stabilized by adjacent binding sites in the CS and the C-tail of the ␤4 subunit that interact with the plakin domain of plectin (9, 10). In type I HDs, the interaction of ␤4 with plectin is further reinfor...

T1736 is a novel phosphorylation site on the integrin β4 subunit that is phosphorylated downstream of protein kinase C and EGF receptor activation and is a substrate for protein kinase D1 in vitro and in cells. It contributes to the regulation of HD dynamics through modulating the association of β4 with plectin.

The rat mAb 33A10 recognizes an antigen in a variety of mouse epithelial tissues. In this study, we investigated in detail the expression pattern of the 33A10-defined antigen in the hair follicle. We show that 33A10 reactivity is confined to the most differentiated keratinocytes of the outer root sheath (ORS), the companion layer (CL), and to cells of the sebaceous gland duct. In vitro, the 33A10-defined antigen is expressed in keratinocytes derived from the ORS and accumulates on induction of differentiation. Using microarray analysis and transient transfection approaches, we established that the 33A10-defined antigen is the orphan protein, Placenta-expressed transcript (Plet)-1. Biochemical data indicated that Plet-1 is a glycosylphosphatidylinositol-anchored glycoprotein with N-linked carbohydrates in addition to other posttranslational modifications. Although silencing of Plet-1 expression using stable RNA interference in ORS keratinocytes decreased cellular migration, it increased adhesion to collagens I and IV. Immunohistochemical analysis showed that Plet-1 was primarily localized at the leading edge of epidermal wounds, where keratinocytes contacted the eschar. The restricted localization in both differentiated ORS and CL cells contacting the hair fiber and epidermal wounds suggests a role for the Plet-1 protein in regulating the interaction of keratinocytes with inert tissues.

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