TY - JOUR
T1 - Phosphorylation of the norepinephrine transporter at threonine 258 and serine 259 is linked to protein kinase C-mediated transporter internalization
AU - Jayanthi, Lankupalle D
AU - Annamalai, Balasubramaniam
AU - Samuvel, Devadoss J
AU - Gether, Ulrik
AU - Ramamoorthy, Sammanda
PY - 2006
Y1 - 2006
N2 - Recently, we have demonstrated the phosphorylation- and lipid raft-mediated internalization of the native norepinephrine transporter (NET) following protein kinase C (PKC) activation (Jayanthi, L. D., Samuvel, D. J., and Ramamoorthy, S. (2004) J. Biol. Chem. 279, 19315-19326). Here we tested an hypothesis that PKC-mediated phosphorylation of NET is required for transporter internalization. Phosphoamino acid analysis of 32P-labeled native NETs from rat placental trophoblasts and heterologously expressed wild type human NET (WT-hNET) from human placental trophoblast cells revealed that the phorbol ester (beta-PMA)-induced phosphorylation of NET occurs on serine and threonine residues. Beta-PMA treatment inhibited NE transport, reduced plasma membrane hNET levels, and stimulated hNET phosphorylation in human placental trophoblast cells expressing the WT-hNET. Substance P-mediated activation of the G alpha(q)-coupled human neurokinin 1 (hNK-1) receptor coexpressed with the WT-hNET produced effects similar to beta-PMA via PKC stimulation. In striking contrast, an hNET double mutant harboring T258A and S259A failed to show NE uptake inhibition and plasma membrane redistribution by beta-PMA or SP. Most interestingly, the plasma membrane insertion of the WT-hNET and hNET double mutant were not affected by beta-PMA. Although the WT-hNET showed increased endocytosis and redistribution from caveolin-rich plasma membrane domains following beta-PMA treatment, the hNET double mutant was completely resistant to these PKC-mediated effects. In addition, the PKC-induced phosphorylation of hNET double mutant was significantly reduced. In the absence of T258A and S259A mutations, alanine substitution of all other potential phosphosites within the hNET did not block PKC-induced phosphorylation and down-regulation. These results suggest that Thr-258 and Ser-259 serve as a PKC-specific phospho-acceptor site and that phosphorylation of this motif is linked to PKC-induced NET internalization.
AB - Recently, we have demonstrated the phosphorylation- and lipid raft-mediated internalization of the native norepinephrine transporter (NET) following protein kinase C (PKC) activation (Jayanthi, L. D., Samuvel, D. J., and Ramamoorthy, S. (2004) J. Biol. Chem. 279, 19315-19326). Here we tested an hypothesis that PKC-mediated phosphorylation of NET is required for transporter internalization. Phosphoamino acid analysis of 32P-labeled native NETs from rat placental trophoblasts and heterologously expressed wild type human NET (WT-hNET) from human placental trophoblast cells revealed that the phorbol ester (beta-PMA)-induced phosphorylation of NET occurs on serine and threonine residues. Beta-PMA treatment inhibited NE transport, reduced plasma membrane hNET levels, and stimulated hNET phosphorylation in human placental trophoblast cells expressing the WT-hNET. Substance P-mediated activation of the G alpha(q)-coupled human neurokinin 1 (hNK-1) receptor coexpressed with the WT-hNET produced effects similar to beta-PMA via PKC stimulation. In striking contrast, an hNET double mutant harboring T258A and S259A failed to show NE uptake inhibition and plasma membrane redistribution by beta-PMA or SP. Most interestingly, the plasma membrane insertion of the WT-hNET and hNET double mutant were not affected by beta-PMA. Although the WT-hNET showed increased endocytosis and redistribution from caveolin-rich plasma membrane domains following beta-PMA treatment, the hNET double mutant was completely resistant to these PKC-mediated effects. In addition, the PKC-induced phosphorylation of hNET double mutant was significantly reduced. In the absence of T258A and S259A mutations, alanine substitution of all other potential phosphosites within the hNET did not block PKC-induced phosphorylation and down-regulation. These results suggest that Thr-258 and Ser-259 serve as a PKC-specific phospho-acceptor site and that phosphorylation of this motif is linked to PKC-induced NET internalization.
KW - Alanine
KW - Amino Acid Motifs
KW - Amino Acid Sequence
KW - Amino Acid Substitution
KW - Animals
KW - Cell Line
KW - Cells, Cultured
KW - Down-Regulation
KW - Enzyme Activation
KW - Humans
KW - Molecular Sequence Data
KW - Mutagenesis, Site-Directed
KW - Norepinephrine Plasma Membrane Transport Proteins
KW - Phosphorylation
KW - Protein Kinase C
KW - Rats
KW - Serine
KW - Threonine
U2 - 10.1074/jbc.M601156200
DO - 10.1074/jbc.M601156200
M3 - Journal article
C2 - 16740633
VL - 281
SP - 23326
EP - 23340
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 33
ER -