Interaction of plakophilins with desmoplakin and intermediate filament proteins: an in vitro analysis.
Plakophilin 1 and 2 (PKP1, PKP2) are members of the arm-repeat protein family. They are both constitutively expressed in most vertebrate cells, in two splice forms named a and b, and display a remarkable dual location: they occur in the nuclei of cells and, in epithelial cells, at the plasma membrane within the desmosomal plaques. We have shown by solid phase-binding assays that both PKP1a and PKP2a bind to intermediate filament (IF) proteins, in particular to cytokeratins (CKs) from epidermal as well as simple epithelial cells and, to some extent, to vimentin. In line with this we show that recombinant PKP1a binds strongly to IFs assembled in vitro from CKs 8/18, 5/14, vimentin or desmin and integrates them into thick (up to 120 nm in diameter) IF bundles extending for several microm. The basic amino-terminal, non-arm-repeat domain of PKP1a is necessary and sufficient for this specific interaction as shown by blot overlay and centrifugation experiments. In particular, the binding of PKP1a to IF proteins is saturable at an approximately equimolar ratio. In extracts from HaCaT cells, distinct soluble complexes containing PKP1a and desmoplakin I (DPI) have been identified by co-immunoprecipitation and sucrose density fractionation. The significance of these interactions of PKP1a with IF proteins on the one hand and desmoplakin on the other is discussed in relation to the fact that PKP1a is not bound - and does not bind - to extended IFs in vivo. We postulate that (1) effective cellular regulatory mechanisms exist that prevent plakophilins from unscheduled IF-binding, and (2) specific desmoplakin interactions with either PKP1, PKP2 or PKP3, or combinations thereof, are involved in the selective recruitment of plakophilins to the desmosomal plaques.
Hofmann
I
I
Division of Cell Biology/A0100 and Protein Analysis Facility/R0800, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. i.hofmann@dkfz-heidelberg.de
Mertens
C
C
Brettel
M
M
Nimmrich
V
V
Schnölzer
M
M
Herrmann
H
H
D002457:Cell Extracts / Q000276:immunology; D002498:Centrifugation; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D051180:Desmoplakins; D003896:Desmosomes / Q000378:metabolism; D066298:In Vitro Techniques; D007381:Intermediate Filament Proteins / Q000378:metabolism* / Q000648:ultrastructure; D008854:Microscopy, Electron; D051191:Plakophilins; D011506:Proteins / Q000378:metabolism*; D011869:Radioligand Assay; D020349:Surface Plasmon Resonance
D002457:Cell Extracts; D003598:Cytoskeletal Proteins; D051180:Desmoplakins; D007381:Intermediate Filament Proteins; C495337:PKP2 protein, human; C495339:PKP3 protein, human; D051191:Plakophilins; D011506:Proteins