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  • Nuclear focal adhesion kinase induces APC/C activator protein CDH1-mediated cyclin-dependent kinase 4/6 degradation and inhibits melanoma proliferation.

Nuclear focal adhesion kinase induces APC/C activator protein CDH1-mediated cyclin-dependent kinase 4/6 degradation and inhibits melanoma proliferation.

The Journal of biological chemistry (2022-05-08)
James M Murphy, Kyuho Jeong, Eun-Young Erin Ahn, Ssang-Taek Steve Lim
ABSTRACT

Dysregulation of cyclin-dependent kinases (CDKs) can promote unchecked cell proliferation and cancer progression. Although focal adhesion kinase (FAK) contributes to regulating cell cycle progression, the exact molecular mechanism remains unclear. Here, we found that FAK plays a key role in cell cycle progression potentially through regulation of CDK4/6 protein expression. We show that FAK inhibition increased its nuclear localization and induced G1 arrest in B16F10 melanoma cells. Mechanistically, we demonstrate nuclear FAK associated with CDK4/6 and promoted their ubiquitination and proteasomal degradation through recruitment of CDC homolog 1 (CDH1), an activator and substrate recognition subunit of the anaphase-promoting complex/cyclosome E3 ligase complex. We found the FAK N-terminal FERM domain acts as a scaffold to bring CDK4/6 and CDH1 within close proximity. However, overexpression of nonnuclear-localizing mutant FAK FERM failed to function as a scaffold for CDK4/6 and CDH1. Furthermore, shRNA knockdown of CDH1 increased CDK4/6 protein expression and blocked FAK inhibitor-induced reduction of CDK4/6 in B16F10 cells. In vivo, we show that pharmacological FAK inhibition reduced B16F10 tumor size, correlating with increased FAK nuclear localization and decreased CDK4/6 expression compared with vehicle controls. In patient-matched healthy skin and melanoma biopsies, we found FAK was mostly inactive and nuclear localized in healthy skin, whereas melanoma lesions showed increased active cytoplasmic FAK and elevated CDK4 expression. Taken together, our data demonstrate that FAK inhibition blocks tumor proliferation by inducing G1 arrest, in part through decreased CDK4/6 protein stability by nuclear FAK.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Anti-FAK Antibody, clone 4.47, clone 4.47, Upstate®, from mouse
Sigma-Aldrich
Anti-Ubiquitin Mouse mAb (FK2), liquid, clone FK2, Calbiochem®
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Monoclonal ANTI-FLAG® M2 antibody produced in mouse, clone M2, purified immunoglobulin (Purified IgG1 subclass), buffered aqueous solution (10 mM sodium phosphate, 150 mM NaCl, pH 7.4, containing 0.02% sodium azide)
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Anti-GST Tag Antibody, Upstate®, from mouse
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Rabbit anti-PCNA Antibody, Affinity Purified, Powered by Bethyl Laboratories, Inc.
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Anti-Glyceraldehyde-3-Phosphate Dehydrogenase Antibody, clone 6C5, clone 6C5, Chemicon®, from mouse