Nesprin-3, a novel outer nuclear membrane protein, associates with the cytoskeletal linker protein plectin

Despite their importance in cell biology, the mechanisms that maintain the nucleus in its proper position in the cell are not well understood. This is primarily the result of an incomplete knowledge of the proteins in the outer nuclear membrane (ONM) that are able to associate with the different cytoskeletal systems. Two related ONM proteins, nuclear envelope spectrin repeat (nesprin)–1 and –2, are known to make direct connections with the actin cytoskeleton through their NH2-terminal actin-binding domain (ABD). We have now isolated a third member of the nesprin family that lacks an ABD and instead binds to the plakin family member plectin, which can associate with the intermediate filament (IF) system. Overexpression of nesprin-3 results in a dramatic recruitment of plectin to the nuclear perimeter, which is where these two molecules are colocalized with both keratin-6 and -14. Importantly, plectin binds to the integrin α6β4 at the cell surface and to nesprin-3 at the ONM in keratinocytes, suggesting that there is a continuous connection between the nucleus and the extracellular matrix through the IF cytoskeleton

Specificity of Binding of the Plectin Actin-binding Domain to β4 Integrin

Plectin is a major component of the cytoskeleton and links the intermediate filament system to hemidesmosomes by binding to the integrin β4 subunit. Previously, a binding site for β4 was mapped on the actin-binding domain (ABD) of plectin and binding of β4 and F-actin to plectin was shown to be mutually exclusive. Here we show that only the ABDs of plectin and dystonin bind to β4, whereas those of other actin-binding proteins do not. Mutations of the ABD of plectin-1C show that Q131, R138, and N149 are critical for tight binding of the ABD to β4. These residues form a small cavity, occupied by a well-ordered water molecule in the crystal structure. The β4 binding pocket partly overlaps with the actin-binding sequence 2 (ABS2), previously shown to be essential for actin binding. Therefore, steric interference may render binding of β4 and F-actin to plectin mutually exclusive. Finally, we provide evidence indicating that the residues preceding the ABD in plectin-1A and -1C, although unable to mediate binding to β4 themselves, modulate the binding activity of the ABD for β4. These studies demonstrate the unique property of the plectin-ABD to bind to both F-actin and β4, and explain why several other ABD-containing proteins that are expressed in basal keratinocytes are not recruited into hemidesmosomes

Serine Phosphorylation of the Integrin β4 Subunit Is Necessary for Epidermal Growth Factor Receptor–induced Hemidesmosome Disruption

Hemidesmosomes (HDs) are multiprotein adhesion complexes that promote attachment of epithelial cells to the basement membrane. The binding of α6β4 to plectin plays a central role in their assembly. We have defined three regions on β4 that together harbor all the serine and threonine phosphorylation sites and show that three serines (S1356, S1360, and S1364), previously implicated in HD regulation, prevent the interaction of β4 with the plectin actin-binding domain when phosphorylated. We have also established that epidermal growth factor receptor activation, which is known to function upstream of HD disassembly, results in the phosphorylation of only one or more of these three residues and the partial disassembly of HDs in keratinocytes. Additionally, we show that S1360 and S1364 of β4 are the only residues phosphorylated by PKC and PKA in cells, respectively. Taken together, our studies indicate that multiple kinases act in concert to breakdown the structural integrity of HDs in keratinocytes, which is primarily achieved through the phosphorylation of S1356, S1360, and S1364 on the β4 subunit