PKC and PKA phosphorylation affect the subcellular localization of claudin-1 in melanoma cells.
Cytoplasmic expression of claudin-1 in metastatic melanoma cells correlates to increased migration, and increased secretion of MMP-2 in a PKC dependent manner, whereas claudin-1 nuclear expression is found in benign nevi. Melanoma cells were transfected with a vector expressing CLDN-1 fused to a nuclear localization signal (NLS). Despite significant nuclear localization of claudin-1, there was still transport of claudin-1 to the cytoplasm. Phorbol ester treatment of cells transfected with NLS-claudin-1 resulted in an exclusion of claudin-1 from the nucleus, despite the NLS. To ascertain whether PKC or PKA were involved in this translocation, we mutated the putative phosphorylation sites within the protein. We found that mutating the PKC phosphorylation sites to mimic a non-phosphorylated state did not cause a shift of claudin-1 to the nucleus of the cells, but mutating the PKA sites did. Mutations of either site to mimic constitutive phosphorylation resulted in cytoplasmic claudin-1 expression. Stable claudin-1 transfectants containing non-phosphorylatable PKA sites exhibited decreased motility. These data imply that subcellular localization of claudin-1 can be controlled by phosphorylation, dicating effects on metastatic capacity.
French
Amanda D
AD
Laboratory of Immunology, National Institute on Aging, Baltimore, MD 21124, USA.
Fiori
Jennifer L
JL
Camilli
Tura C
TC
Leotlela
Poloko D
PD
O'Connell
Michael P
MP
Frank
Brittany P
BP
Subaran
Sarah
S
Indig
Fred E
FE
Taub
Dennis D
DD
Weeraratna
Ashani T
AT
D001692:Biological Transport / Q000235:genetics; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D062445:Claudin-1; D003198:Computer Simulation; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D005822:Genetic Vectors; D006801:Humans; D007150:Immunohistochemistry; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D008545:Melanoma / Q000378:metabolism / Q000473:pathology*; D008565:Membrane Proteins / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009361:Neoplasm Invasiveness; D009362:Neoplasm Metastasis; D019913:Nuclear Localization Signals / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
C568108:CLDN1 protein, human; D062445:Claudin-1; D008565:Membrane Proteins; D019913:Nuclear Localization Signals; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D020778:Matrix Metalloproteinase 2; D013755:Tetradecanoylphorbol Acetate