The detachment of epithelial cells from the basal matrix during wound healing and differentiation of keratinocytes requires the disassembly of the hemidesmosomal multiprotein adhesion complex. Integrin ␣64-plectin interaction plays a major role in the formation of hemidesmosomes, and thus the mechanisms regulating this interaction should be critical also for the disassembly process. Here we show that a particular plectin isoform (1a) interacts with the Ca 2؉ -sensing protein calmodulin in a Ca 2؉ -dependent manner. As a result of this interaction, binding of the hemidesmosome-associated plectin isoform 1a to integrin 4 is substantially diminished. Calmodulin-binding inhibits also the interaction of plectin with F-actin. Further, we found that, during Ca 2؉ -induced keratinocyte differentiation, plectin 1a is first relocated within the cell and later down-regulated, suggesting that Ca 2؉ affects the fate of plectin 1a upon its release from hemidesmosomes. We propose a novel model for the disassembly of hemidesmosomes during keratinocyte differentiation, where both, binding of calmodulin to plectin 1a and phosphorylation of integrin 4 by protein kinases, are required for disruption of the integrin ␣64-plectin complex.Basal cell layer keratinocytes of the skin are firmly attached to the underlying basement membrane through hemidesmosomes (HDs), 2 protein complexes that mediate stable anchoring by providing a tight link between the intracellular intermediate filament network (IF) system and the extracellular matrix (1, 2). Plectin, a universal and functionally versatile cytolinker protein, has been implicated in HD functions since early on (3, 4). Binding of plectin to the 4 subunit of integrin ␣64 turned out to be a critical step in the formation of HDs, and various regions of plectin have been shown to be involved in this interaction. One of them is a canonical actin-binding domain (ABD) of the tandem calponin homology (CH) domain type (5-7), which binds to the second fibronectin type III (FnIII) domain of the 4 subunit. Residues crucial for this binding are clustered on the surface of the first N-terminal calponin homology domain. They partially overlap with one of the essential F-actin-binding sequences, providing a structural basis for the competitive nature of plectin binding to integrin 4 and F-actin (2,8). A second integrin 4-binding site, located in the plakin domain of plectin (9), interacts with sequences downstream of the second FnIII domain of integrin 4 (10). In addition, interactions of the C-terminal repeat domain R6 of plectin with the segment connecting the second and third FnIII domains of integrin 4 (C-segment), and with integrin 4 tail domain, have been reported (9).In accordance with plectin being a prominent constituent of HDs, abnormalities in its expression in humans lead to epidermolysis bullosa simplex associated with muscular dystrophy, manifesting as severe skin blistering and muscle disorders (for review see Ref. 11); an autosomal dominant form of the disease (epidermolysis ...