Identification of tyrosines in the putative regulatory site of the Ca2+ channel TRPV6.
In HEK293 cells, transfected with the Ca2+ channel protein TRPV6, Ca2+ influx is increased and TRPV6 is tyrosine phosphorylated following addition of the tyrosine phosphatase inhibitor N,N-dimethyl-hydroxamido hydroxovanadate to cells. This effect of DMHV is enhanced by co-transfection of cells with the tyrosine kinase Src and the tyrosine phosphatase 1B. It is abolished when cells had been treated with PP1, an inhibitor of Src family tyrosine kinases. PTP1B interacts with the N-terminal domain of TRPV6 within a region of amino acids 1-191 as shown by co-immunoprecipitation, bimolecular fluorescence complementation and the yeast 2-hybrid system. Point mutation of both tyrosines 161 and 162 in the TRPV6 protein abolishes the DMHV-effect on Ca2+ influx and tyrosine phosphorylation by Src. Single mutations of Y161 or Y162 shows that each of both tyrosines alone is sufficient for the DMHV-effect. We conclude that phosphorylation/dephosphorylation of tyrosines in position 161 and 162 is essential for regulation of Ca2+ influx through TRPV6 Ca2+ channels in HEK293 cells.
Sternfeld
Lutz
L
Physiological Institute, University of Saarland, 66421 Homburg (Saar), Germany.
Anderie
Ines
I
Schmid
Andreas
A
Al-Shaldi
Hajaj
H
Krause
Elmar
E
Magg
Thomas
T
Schreiner
Dietmar
D
Hofer
Hans-Werner
HW
Schulz
Irene
I
D000595:Amino Acid Sequence; D015220:Calcium Channels / Q000737:chemistry* / Q000235:genetics; D002478:Cells, Cultured; D006801:Humans; D017354:Point Mutation; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D050916:TRPV Cation Channels / Q000737:chemistry* / Q000235:genetics; D014162:Transfection; D020798:Two-Hybrid System Techniques; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology; D019061:src-Family Kinases / Q000502:physiology
D015220:Calcium Channels; D050916:TRPV Cation Channels; C422827:TRPV6 protein, human; C402001:bis(N,N-dimethylhydroxamido)hydroxooxovanadate; D014638:Vanadates; D014443:Tyrosine; D019061:src-Family Kinases; D017027:Protein Tyrosine Phosphatases