Inhibition of Protein-tyrosine Phosphatase Stimulates the Dynamin-dependent Endocytosis of ROMK1

Abstract

We have previously shown that inhibiting proteintyrosine kinase increased whereas inhibiting proteintyrosine 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 phosphorylationinduced 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 dynamindependent internalization of the channel. ROMK11 is located in the apical membrane of the cortical collecting duct (CCD) and is generally believed to be a key component of the native small conductance K ϩ (SK) channel (2-6). The SK channels are the major contributors to the apical K ϩ conductance and are responsible for K ϩ secretion (4, 7). One important factor for regulating K ϩ secretion is the dietary K ϩ intake; a high K ϩ intake increases whereas a low K ϩ intake decreases K ϩ secretion (7). The low K ϩ intake-induced decrease in K ϩ secretion is at least partially achieved by reducing the number of SK channels in the apical membrane of the CCD (8).Our preceding experiments strongly indicated that the low K ϩ intake-induced decrease in SK channel number was mediated by protein-tyrosine kinase (PTK). This conclusion is supported by the observation that inhibition of PTK increased the number of the SK channels in the apical membrane of the CCD from rats on a K ϩ -deficient diet (8). In contrast, inhibition of PTP decreased the number of SK channels in the CCD from rats on a high K ϩ diet (9). Because the effect of inhibiting PTP on channel activity was blocked by 20% sucrose, we speculated that inhibiting PTP increases the endocytosis of the SK channels whereas inhibiting PTK augments the exocytosis of the SK channels into the cell membrane. This notion is supported by observations that inhibiting PTP with PAO reduced whereas inhibiting PTK with herbimycin A increased the membrane location of ROMK1 in oocytes injected with GF...

Receptor tyrosine kinases positively regulate BACE activity and Amyloid-β production through enhancing BACE internalization

“…In addition, overexpression of c-Src itself could mimic the effects of RTK activation, indicating that c-Src is not only necessary but also sufficient for BACE internalization. It is known that c-Src mediates internalization of membrane proteins by phosphorylating the internalized protein or components of the internalization machinery [20][21][22]24]. Our results suggest that in this case c-Src may phosphorylate components of the internalization machinery because no tyrosine residue was found in the cytosolic tail of BACE [16,21,23,24,62,63].…”

“…Upon activation, RTKs undergo phosporylation at specific tyrosine sites and then bind with SH2 (Src homology region) or PTB (phosphotyrosine binding) domain-containing proteins to activate downstream effectors such as c-Src, MAPK, and PKC [19]. Particularly, c-Src has been demonstrated to regulate the internalization of membrane proteins via different mechanisms [20][21][22][23][24], implicating a means for RTK-mediated regulation of other membrane proteins. RTK internalization is required for their normal signaling.…”

Romk1 Knockout Mice Do Not Produce Bartter Phenotype but Exhibit Impaired K Excretion

“…Although both ROMK1 and ROMK2 transcripts are expressed in the CNT and CCD (9), only the ROMK1 isoform, which is exclusively expressed in these nephron segments, is regulated by PTK (31,32). PTK-stimulated endocytosis of ROMK1 in oocytes and HEK cells requires phosphorylation of the C-terminal consensus motif (Tyr-337 in ROMK1) (31). Because all ROMK iso- forms (1-3) have a C-terminal PTK phosphorylation motif, the reason for the isoform specificity of regulation by PTK likely involves the unique ROMK1 NH 2 terminus.…”

“…Endocytosis of romk channels is a dynamin-dependent process using clathrin-coated pits (30). Although both ROMK1 and ROMK2 transcripts are expressed in the CNT and CCD (9), only the ROMK1 isoform, which is exclusively expressed in these nephron segments, is regulated by PTK (31,32). PTK-stimulated endocytosis of ROMK1 in oocytes and HEK cells requires phosphorylation of the C-terminal consensus motif (Tyr-337 in ROMK1) (31).…”

Src-family protein tyrosine kinase phosphorylates WNK4 and modulates its inhibitory effect on KCNJ1 (ROMK)

“…However, the stimulatory effect of SGK1 on WNK4's phosphorylation was absent in the presence of high SFK activity (12). A large body of evidence has demonstrated that SFK plays an important role in inhibiting ROMK channels in the CCD (24)(25)(26)(27)(28)(29). We have previously demonstrated that SFK phosphorylates the ROMK1 channel at Tyr 337 (14) thereby facilitating internalization of ROMK channels (24).…”