- Email: [email protected]
Kinases, cell volume, and the regulation of epithelial transport
ROLE OF PDZ ROTEINS NHERF AND E3KARP IN CYCLIC NUCLEOTIDE REGULATION OF NHE3 Lamprecht G., Cha B-Y., Nadarajah J., Kwon-Lee W., deJonge H. and Donowitz M. GI Division, Johns Hopkins Un. Sch of Med; Balt., MD and Dept Biochem, Un Rotterdam, Rotterdam, NE. Regulation of the epithelial brush border Na/H exchanger (NHE) NHE3 has been studied in intact tissue, polarized epithelial cells and when expressed in a fibroblast model, PS I20 cells. In intact ileum, agents which elevate CAMP inhibit NHE3, including secretin, VIP, and cholera toxin; while agents which elevate cGMP also inhibit NHE3. including guanylin and heat stable E. coli enterotoxin. In PSl20/NHE3 cells, neither CAMP nor cGMP inhibit NHE3, while another NHE (trout RBC BNHE) is inhibited in the same cells by elevation of CAMP. We have shown that the explanation for the lack of cyclic nucleotide effects on NHE3 in PSI20 cells is due to lack of members of the Regulatory Factor (RF) gene family, NHERF (Na/H exchange regulatory factor) and E3KARP (NHE3 kinase A regulatory protein). These proteins when expressed in PSI20 cells reconstitute CAMP inhibition of NHE3 by a mechanism which involves both a decrease in Vmax and a decrease in K’(H+)i. These proteins contain two PDZ domains and it is the second PDZ domain plus the Cterminus which binds NHE3. The part of NHE3 binding NHERF/E3KARP is between aa 585-71 I. In addition to binding NHE3, NHERF and E3KARP bind ezrin, a cytoskeletal linking protein which is present in PS 120 cells. The N-terminus of ezrin binds to the C-terminal 23-30 aa of NHERF/E3KARP. Truncating NHERF/E3KARP C-terminus to remove the part of the protein which binds ezrin and expressing the truncations in PS I20/NHE3 cells eliminates CAMP inhibition of NHE3. Ezrin is a low affinity AKAP. CAMP inhibits NHE3 by inducing PKA II induced phosphorylation of NHE3. In PS 120 cells, CAMP does not phosphorylate NHE3 unless either E3KARP or NHERF are transfected into the cells. By confocal microscopy, NHERF and E3KARP are in the plasma membrane and cytosol in PSI20 cells and co-localize with NHE3 in the plasma membrane. Neither NHE3 nor E3KARP/NHERF change location when exposed to CAMP. Similarly to the effect of CAMP, cGMP inhibition of NHE3 can be reconstituted by transfecting E3KARP, however, the effect is specific in that NHERF does not cause similar reconstitution. These studied required infection of the PS 120 cells with replication deficient adenovirus containing cGMP kinase II which is absent from PS I20 cells. cGMP kinase binds E3KARP but not NHERF. Conclusion: The RF proteins are required for CAMP and cGMP inhibition of NHE3 and act as scaffolds to anchor NHE3 to the cytoskeleton and to protein kinases.
KINASES, CELL VOLUME, AND THE REGULATION OF EPITHELIAL TRANSPORT Badawi C.‘, Gamper N.‘, Huber S.‘, Fillon S.‘, Lepple-Wienhues A.‘, Waldegger S.‘, Wagner C.A.‘, Cohen P.‘, Moschen I.‘, Setiawan I.‘, Feng Y.‘, Stegen C.‘, Friedrich B.‘, Broer S.‘, Szabo I.‘, Siemen D.‘, Gulbins E.’ and Lang F.’ Departments for Physiology’ and Biochemistry*, Universities of Tubingen and Dundee Ample pharmacological evidence points to a role of kinases in the regulation of cell volume. Given the limited selectivity of most inhibitors, however, the specific molecules involved have remained largely elusive. In this presentation two types of kinases are described which are involved in the regulation of cell volume sensitive transport systems: Swelling of Jurkat T lymphocytes leads to activation of the tyrosine kinase Ick”, which in turn activates the cell volume regulatory Cl-channel ORCC. Pharmacological inhibition (herbimycin, lavendustin, genistein) and genetic deficiency of Lck’” abolish activation of ORCC and regulatory cell volume decrease (RVD) while retransfection of Lcksh into Lck’“deficient cells restores activation of ORCC and RVD. Purified Lcks6 stimulates ORCC in excised patches pointing to direct interaction with the channel. Surprisingly, though, L&6 may mediate tyrosine-phosphorylation and inhibition of Kvl.3, the K’-channel considered important for RVD in lymphocytes. Lck” can be activated by ceramide, which is released from sphingomyelinase, an enzyme, however, apparently not activated by cell swelling. The search for cell volume regulated genes lead to the discovery of the human serum and glucocorticoid dependent serineithreonine kinase hsgkl. Transcription of hsgkl is markedly and rapidly upregulated by osmotic and isotonic cell shrinkage. Coexpression of hsgkl with renal epithelial Naf-channel ENaC or with Na+,KC,2K+-cotransporter NKCC into Xenopus oocytes accelerates insertion of the transport proteins into the cell membrane and thus stimulates channel or transport activity. Surprisingly, though, hsgkl similarly activates voltage gated K+-channels, as apparent from patch clamp in hsgkl overexpressing human embryonic kidney (HEK) cells and voltage clamp in Xenopus oocytes. The effects of hsgk I are mimicked by sgk2 and sgk3, isoforms of hsgk I. The above mechanisms may participate in the regulation of epithelial transport. Lck” is not expressed in epithelial tissues but pharmacological inhibition of tyrosine kinases interferes with RVD in those cells. Thus, a similar kinase may mediate RVD and transport regulation in epithelia. hsgkl is expressed in a variety of epithelial tissues including intestine and kidney. Its transcription is upregulated by mineralocorticoids and hsgkl has thus been suggested to mediate the early effects of aldosterone. As sgk2 and sgk3 are similarly transcribed in pancreatic tissue, liver and kidney, they could similarly participate in transport regulation.
KINASES, CELL VOLUME, AND THE REGULATION OF EPITHELIAL TRANSPORT Badawi C.‘, Gamper N.‘, Huber S.‘, Fillon S.‘, Lepple-Wienhues A.‘, Waldegger S.‘, Wagner C.A.‘, Cohen P.‘, Moschen I.‘, Setiawan I.‘, Feng Y.‘, Stegen C.‘, Friedrich B.‘, Broer S.‘, Szabo I.‘, Siemen D.‘, Gulbins E.’ and Lang F.’ Departments for Physiology’ and Biochemistry*, Universities of Tubingen and Dundee Ample pharmacological evidence points to a role of kinases in the regulation of cell volume. Given the limited selectivity of most inhibitors, however, the specific molecules involved have remained largely elusive. In this presentation two types of kinases are described which are involved in the regulation of cell volume sensitive transport systems: Swelling of Jurkat T lymphocytes leads to activation of the tyrosine kinase Ick”, which in turn activates the cell volume regulatory Cl-channel ORCC. Pharmacological inhibition (herbimycin, lavendustin, genistein) and genetic deficiency of Lck’” abolish activation of ORCC and regulatory cell volume decrease (RVD) while retransfection of Lcksh into Lck’“deficient cells restores activation of ORCC and RVD. Purified Lcks6 stimulates ORCC in excised patches pointing to direct interaction with the channel. Surprisingly, though, L&6 may mediate tyrosine-phosphorylation and inhibition of Kvl.3, the K’-channel considered important for RVD in lymphocytes. Lck” can be activated by ceramide, which is released from sphingomyelinase, an enzyme, however, apparently not activated by cell swelling. The search for cell volume regulated genes lead to the discovery of the human serum and glucocorticoid dependent serineithreonine kinase hsgkl. Transcription of hsgkl is markedly and rapidly upregulated by osmotic and isotonic cell shrinkage. Coexpression of hsgkl with renal epithelial Naf-channel ENaC or with Na+,KC,2K+-cotransporter NKCC into Xenopus oocytes accelerates insertion of the transport proteins into the cell membrane and thus stimulates channel or transport activity. Surprisingly, though, hsgkl similarly activates voltage gated K+-channels, as apparent from patch clamp in hsgkl overexpressing human embryonic kidney (HEK) cells and voltage clamp in Xenopus oocytes. The effects of hsgk I are mimicked by sgk2 and sgk3, isoforms of hsgk I. The above mechanisms may participate in the regulation of epithelial transport. Lck” is not expressed in epithelial tissues but pharmacological inhibition of tyrosine kinases interferes with RVD in those cells. Thus, a similar kinase may mediate RVD and transport regulation in epithelia. hsgkl is expressed in a variety of epithelial tissues including intestine and kidney. Its transcription is upregulated by mineralocorticoids and hsgkl has thus been suggested to mediate the early effects of aldosterone. As sgk2 and sgk3 are similarly transcribed in pancreatic tissue, liver and kidney, they could similarly participate in transport regulation.