Focal adhesion kinase pp125FAK interacts with the large conductance calcium-activated hSlo potassium channel in human osteoblasts: potential role in mechanotransduction.
Molecular events of mechanotransduction in osteoblasts are poorly defined. We show that the mechanosensitive BK channels open and recruit the focal adhesion kinase FAK in osteoblasts on hypotonic shock. This could convert mechanical signals in biochemical events, leading to osteoblast activation.


BACKGROUND
Mechanical strains applied to the skeleton influence bone remodeling and architecture mainly through the osteoblast lineage. The molecular mechanisms involved in osteoblastic mechanotransduction include opening of mechanosensitive cation channels and the activation of protein tyrosine kinases, notably FAK, but their interplay remains poorly characterized. The large conductance K+ channel (BK) seems likely as a bone mechanoreceptor candidate because of its high expression in osteoblasts and its ability to open in response to membrane stretch or hypotonic shock. Propagation of the signals issued from the mechanosensitivity of BK channels inside the cell likely implies complex interactions with molecular partners involved in mechanotransduction, notably FAK.


METHODS
Interaction of FAK with the C terminus of the hSlo alpha-subunit of BK was investigated using the yeast two-hybrid system as well as immunofluorescence microscopy and coimmunoprecipitation experiments with a rabbit anti-hslo antibody on MG63 and CAL72 human osteosarcoma cell lines and on normal human osteoblasts. Mapping of the FAK region interacting with hSlo was approached by testing the ability of hSlo to recruit mutated ot truncated FAK proteins.


RESULTS
To the best of our knowledge, we provide the first evidence of the physical association of FAK with the intracellular part of hslo. We show that FAK/hSlo interaction likely takes place through the Pro-1-rich domain situated in the C-terminal region of the kinase. FAK/hSlo association occurs constitutively at a low, but appreciable, level in human osteosarcoma cells and normal human osteoblasts that express endogenous FAK and hSlo. In addition, we found that application of an hypo-osmotic shock to these cells induced a sustained activation of BK channels associated to a marked increase in the recruitment of FAK on hSlo.


CONCLUSIONS
Based on these data, we propose that BK channels might play a triggering role in the signaling cascade induced by mechanical strains in osteoblasts.
Faculté de Médecine, INSERM U364, Nice, France. rezzonic@unice.fr
Roger
R
Rezzonico
Corinne
C
Cayatte
Isabelle
I
Bourget-Ponzio
Georges
G
Romey
Nathalie
N
Belhacene
Agnès
A
Loubat
Stéphane
S
Rocchi
Emmanuel
E
Van Obberghen
Jean-Antoine
JA
Girault
Bernard
B
Rossi
Heidy
H
Schmid-Antomarchi
D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D019070:Cell Lineage; D004594:Electrophysiology; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006801:Humans; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D051036:Large-Conductance Calcium-Activated Potassium Channels; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D010006:Osteoblasts / Q000378:metabolism* / Q000473:pathology; D010957:Plasmids / Q000378:metabolism; D015221:Potassium Channels / Q000737:chemistry; D024681:Potassium Channels, Calcium-Activated / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D015398:Signal Transduction; D020798:Two-Hybrid System Techniques
C494841:KCNMA1 protein, human; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D051036:Large-Conductance Calcium-Activated Potassium Channels; D015221:Potassium Channels; D024681:Potassium Channels, Calcium-Activated; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; D002118:Calcium