PKC putative phosphorylation site Ser235 is required for MIP/AQP0 translocation to the plasma membrane.
OBJECTIVE
To investigate the functional significance of MIP/AQP0 phosphorylation at serine(235).


METHODS
MIP/AQP0 expression and cellular localization was studied in rat lens epithelia explants induced to differentiate by FGF-2. MIP wild type (WT) and MIP (S235A) mutant expression plasmids were constructed and transiently expressed in RK13 cells. Subcellular localization of endogenous MIP in differentiating lens epithelia explants or of transfected MIP expression vectors in RK13 cells was analyzed by immunofluorescence confocal microscopy.


RESULTS
MIP/AQP0 expressed in lens epithelia explants induced to differentiate by FGF-2 localizes to the plasma membrane of elongating cells. However, MIP/AQP0 translocation to the plasma membrane was prevented by inhibiting PKC activity with Go6976, resulting in retention in the cytoplasmic compartment. This effect was specific to MIP/AQP0; localization of AQP1 to the cell membrane was not affected by Go6976. When the consensus PKC phosphorylation site at MIP Ser(235) was mutated to alanine and transiently expressed in transfected RK13 cells, the mutant MIP was retained in the cytoplasmic compartment in contrast to WT MIP that localized to the plasma membrane of the transfected RK13 cells. Colocalization studies indicated that the mutant MIP was retained in the trans-Golgi network.


CONCLUSIONS
Our results indicate that serine(235) is required for proper intracellular transport of MIP/AQP0 from the trans-Golgi network to the plasma membrane. A PKC dependent phosphorylation event involving MIP at serine(235) is most likely involved in this process.
Laboratory of Molecular and Developmental Biology, National Eye Institute, NIH, Bethesda, MD 20892-2510, USA.
Nady
N
Golestaneh
Jianguo
J
Fan
Peggy
P
Zelenka
Ana B
AB
Chepelinsky
D000818:Animals; D051398:Aquaporin 1 / Q000378:metabolism; D020346:Aquaporins / Q000235:genetics / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology*; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism*; D066298:In Vitro Techniques; D007908:Lens, Crystalline / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D014158:Transcription, Genetic / Q000187:drug effects; D021601:trans-Golgi Network / Q000187:drug effects / Q000378:metabolism
D020346:Aquaporins; D005136:Eye Proteins; D008562:Membrane Glycoproteins; D050505:Mutant Proteins; D047428:Protein Kinase Inhibitors; C030328:aquaporin 0; D051398:Aquaporin 1; D010768:Phosphoserine; D011493:Protein Kinase C