REGULATION OF DRUG TRANSPORTER ACTIVITY - Drug Transporters: Molecular Characterization and Role in Drug Disposition - page 522

17.4. PHOSPHORYLATION
Phosphorylation is the covalent attachment of one or several phosphate groups to the
hydroxyl side chains of serine, threonine, or tyrosine on proteins. Most phosphate
on proteins of animal cells is on serine residues, less on threonine, with a very small
amount on tyrosine residues. The phosphorylation process occurs in the cytosol. Phos-
phorylation influences the conformation and charge of the protein, thereby also its
activity (either up or down), cellular location, or association with other proteins. Phos-
phorylation is catalyzed by protein kinases, which move a phosphate group from an
adenosine triphosphate (ATP) molecule to the proteins. Tyrosine kinases phosphory-
late proteins on tyrosine; serine/threonine kinases phosphorylate proteins on serine or
threonine. However, the phosphate groups can also be removed from the protein by a
process called dephosphorylation . This process is catalyzed by protein phosphatases.
The amount of phosphate that is associated with the protein is thus determined by the
relative activities of the kinase and phosphatase. Together, protein kinases and protein
phosphatases act in an exactly opposite fashion to regulate a population of target pro-
teins by controlling their phosphorylation state. Reversible protein phosphorylation is
responsible for regulation of cellular processes as diverse as mobilization of glucose
from glycogen, 19 , 20 prevention of transplant rejection by cyclosporine, 21 and devel-
opment of a cancer form such as chronic myeloid leukemia. 22 , 23 An ion channel, may
be closed when dephosphorylated but open when phosphorylated. Thus, a protein
kinase would be responsible for opening the ion channel, and a protein phosphatase
would be responsible for closing that channel.
Many drug transporters can also be regulated by reversible phosphorylation. It has
been shown 24 that in HEK293 cells stably expressing rat organic cation transporter
rOCT1, stimulation of protein kinase C (PKC) by sn -1,2-dioctanoyl glycerol results
in a significant increase in the transport affinity of rOCT1 for its substrates tetraethy-
lammonium, tetrapenthylammonium, and quinine. Such increase in transport affinity
was accompanied by serine phosphorylation of the transporter. It was therefore pro-
posed that the phosphorylation of rOCT1 by PKC results in conformational changes
at the substrate-binding site.
Mouse organic anion transporter mOAT1 is another example whose transport ac-
tivity is regulated by reversible phosphorylation. It was found 25 that treatment of
mOAT1-expressing LLC-PK1 cells with okadaic acid resulted in an increase in the
phosphorylation of mOAT1. Okadaic acid is a potent inhibitor of protein phosphatase
1 and protein phosphatase 2A, two of the four major serine/threonine protein phos-
phatases in the cytosol of mammalian cells. Okadaic acid readily enters cells, and
numerous studies have demonstrated that it enhances the phosphorylation of many
cellular proteins, presumably by preventing dephosphorylation. It was shown that
okadaic acid-induced phosphorylation of mOAT1 paralleled in time and concen-
tration the decrease of mOAT1-mediated transport of p-aminohippurate (PAH), a
protypical organic anion. Phosphoamino acid analysis indicated that phosphorylation
occurred primarily on serine residues. These results suggest that the increase in serine
phosphorylation of mOAT1 by okadaic acid is, at least in part, responsible for the
okadaic acid-induced decrease in basolateral PAH transport.