Regulation of plakophilin1's dual function in translation and desmosome organization
Final Report Abstract
Plakophilin 1 (PKP1) is a member of the armadillo-family of cell contact proteins with dual functions in intercellular adhesion and cell signalling. When localized in desmosomes, PKP1 enhances intercellular cohesion whereas it stimulates translation and proliferation when localized in the cytoplasm. Thus, it can have a tumour protective function by enhancing contact inhibition of cell growth but also tumour promoting characteristics by increasing proliferation via an induction of protein synthesis. The balance between these two functions correlates with the intracellular localization of PKP1. The aim of the project described here was to unravel the mechanisms that regulate the proportion of the desmosomal versus the cytoplasmic pool of PKP1 thereby modulating its cellular functions. We have shown that PKP1 interacts with the Akt2 but not the closely related Akt1 kinase and to a lesser extent with the S6-kinase (S6K). Both kinases are components of a kinasecascade downstream of the insulin-/IGF1-receptor. Direct interactions were verified in multiple assay systems such as yeast-2-hybrid, GST-pull down, co-immunoprecipitation and Bi-molecular fluorescence complementation. Moreover, these assays showed consistently a preference for the active form of Akt2 suggesting that PKP1 might be a substrate of activated Akt2. Indeed, Akt2 phosphorylated PKP1 in vitro and in cells. Identification of the phosphoryation sites allowed us to analyse the functional consequences of PKP1 phosphorylation directly and independent of other cellular targets of Akt2 by generating a phospho-mimikry mutant as well as a non-phosphorylatable variant. The characterization of these PKP1-mutants showed that the unphosphorylated form localizes exclusively at desmosomes and enhances intercellular cohesion. In contrast, the phospho-mimikry mutant was detected primarily in the cytoplasm where it was protected from degradation. It enhanced protein synthesis and proliferation and was able to induce anchorage independent growth indicating that the expression of this mutant was sufficient to override contact inhibition of cell proliferation. In a physiological context, growth factor signalling via insulin and IGF1 but not EGF activated Akt2 and led to the phosphoryation of PKP1. This correlated with a PKP1 dependent reduction in intercellular adhesion and an increase in proliferation. Our results demonstrate a context dependent regulation of PKP1 that switches its function from enhancing contact inhibition to increasing proliferation and overriding contact inhibition thus conferring a tumour promoting function.
Publications
- Insulin signaling via Akt2 switches plakophilin 1 function from stabilizing cell adhesion to promoting cell proliferation. J Cell Sci. 2013 Apr 15;126(Pt 8):18 32-44
Wolf A, Rietscher K, Glaß M, Hüttelmaier S, Schutkowski M, Ihling C, Sinz A, Wingenfeld A, Mun A, Hatzfeld M
(See online at https://doi.org/10.1242/jcs.118992) - Plakophilins in desmosomal adhesion and signaling. Cell Commun Adhes. 2014 Feb;21(1):25-42
Hatzfeld M, Wolf A, Keil R
(See online at https://doi.org/10.3109/15419061.2013.876017)
