Inhibition of protein-tyrosine phosphatase stimulates the dynamin-dependent endocytosis of ROMK1.
We have previously shown that inhibiting protein-tyrosine kinase increased whereas inhibiting protein-tyrosine phosphatase (PTP) decreased renal outer medullary potassium channel 1 (ROMK1) channel activity (1). We have now used confocal microscopy, the patch clamp technique, and biotin labeling to further examine the role of tyrosine phosphorylation in regulating ROMK1 trafficking. Human embryonic kidney 293 cells were cotransfected with c-Src and green fluorescent protein-ROMK1, which has the same biophysical properties as those of ROMK1. Patch clamp studies have shown that phenylarsine oxide (PAO), an inhibitor of PTP, decreased the activity of ROMK1. Moreover, addition of PAO reduced the cell surface localization of green fluorescent protein-ROMK1 detected by confocal microscopy and diminished the surface ROMK1 density by 65% measured by biotin labeling. Also, PAO treatment significantly increased the phosphorylation of ROMK1. The notion that the effect of PAO is mediated by stimulating tyrosine phosphorylation-induced endocytosis of ROMK1 has also been supported by findings that mutating the tyrosine residue 337 of ROMK1 to alanine abolished the effect of PAO. Finally, the inhibitory effect of PAO on ROMK1 was completely blocked in the cells co-transfected with dominant negative dynamin (dynaminK44A). This indicates that the tyrosine phosphorylation-induced endocytosis of ROMK1 is dynamin-dependent. We conclude that inhibiting PTP increases ROMK1 phosphorylation and results in a dynamin-dependent internalization of the channel.
Sterling
Hyacinth
H
Department of Pharmacology, New York Medical College, Valhalla, New York 10595, USA.
Lin
Dao-Hong
DH
Gu
Rui-Min
RM
Dong
Ke
K
Hebert
Steven C
SC
Wang
Wen-Hui
WH
D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D034281:Dynamins; D004705:Endocytosis / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020558:GTP Phosphohydrolases / Q000502:physiology*; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D015221:Potassium Channels / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D024661:Potassium Channels, Inwardly Rectifying; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D017931:DNA Primers; D004791:Enzyme Inhibitors; C086989:KCNJ1 protein, human; D008164:Luminescent Proteins; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D014443:Tyrosine; D017027:Protein Tyrosine Phosphatases; D020558:GTP Phosphohydrolases; D034281:Dynamins