Abstract
Presenilin 1 and 2 (PS) are critical components of the gamma-secretase complex that cleaves type I transmembrane proteins within their transmembrane domains. This process leads to release of proteolytically processed products from cellular membranes and plays an essential role in signal transduction or vital functions as cell adhesion. Here we studied the function of presenilins in cell-matrix interaction of wild-type and PS knock-out mouse embryonic fibroblasts. We found for PS1(-/-) cells an altered morphology with significantly reduced sizes of focal adhesion sites compared with wild type. Cell force analyses on micropatterned elastomer films revealed PS1(-/-) cell forces to be reduced by 50%. Pharmacological inhibition confirmed this function of gamma-secretase in adhesion site and cell force formation. On the regulatory level, PS1 deficiency was associated with strongly decreased phosphotyrosine levels of focal adhesion site-specific proteins. The reduced tyrosine phosphorylation was caused by a down-regulation of c-Src kinase activity primarily at the level of c-Src transcription. The direct regulatory connection between PS1 and c-Src could be identified with ephrinB2 as PS1 target protein. Overexpression of ephrinB2 cytoplasmic domain resulted in its nuclear translocation with increased levels of c-Src and a full complementation of the PS1(-/-) adhesion and phosphorylation phenotype. Cleavage of full-length EB2 and subsequent intracellular domain translocation depended on PS1 as these processes were only found in WT cells. Therefore, we conclude that gamma-secretase is vital for controlling cell adhesion and force formation by transcriptional regulation of c-Src via ephrinB2 cleavage.
Highlights
PS12 and PS2 are aspartyl proteases forming the active components of the ␥-secretase complex
Because ephrinB1 and ephrinB2 are targets of PS1 and bind to cellular sarcoma protein kinase (c-Src), a protein vitally involved in focal adhesion (FA) formation, it is speculated that Presenilin and 2 (PS) might be an important regulator for cell-cell interaction as well as for switching cell function from a more sessile to a more dynamic, moving phenotype
We identified a significant reduction in FA size upon PS1 deficiency or pharmacological inhibition of ␥-secretase that was associated with a reduction in cell force formation by Ͼ50%
Summary
Cell Culture and Strains—Wild-type mouse embryonic fibroblasts as well as PS1Ϫ/Ϫ, PS2Ϫ/Ϫ, and PS1Ϫ/ϪPS2Ϫ/Ϫ strains of identical genetic background were described earlier and were kindly provided by Dr B. Labeling intensity for various general as well as phosphospecific antibodies against focal adhesion proteins was determined using ImageJ as software (Version 1.36). Immunofluorescence analyses were performed using a laser scanning microscope (LSM 510, Zeiss, Jena, Germany) with filter sets appropriate for simultaneous detection of green (Cy2) and red (Cy3) fluorescent light. 50 g of protein from crude cell extracts were separated on a 10% SDS-polyacrylamide gel and subsequently analyzed by Western blotting. Relative quantitative gene expression was analyzed using Power SYBR Green PCR Master Mix (Applied Biosystems) and specific primers for glyceraldehyde-3-phosphate dehydrogenase as the internal control and c-Src (Eurofins MWG) according to the manufacturer’s instructions. Data were called significantly different for p Ͻ 0.05
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