Activation of Src kinase in skeletal muscle cells by 1, 1,25-(OH(2))-vitamin D(3) correlates with tyrosine phosphorylation of the vitamin D receptor (VDR) and VDR-Src interaction.
The rapid effect of 1 alpha,25(OH(2))-vitamin D(3) [1 alpha, 25(OH(2))D(3)] on tyrosine kinase Src and its relationship to the vitamin D receptor (VDR) was investigated to further characterize the hormone signaling mechanism in chick muscle cells. Exposure of cultured myotubes to 1 alpha,25(OH(2))D(3) caused a time-dependent increase in Src activity, which was evident at 1 min (one-fold) and reached a maximum at 5 min (15-fold). Immunoblotting with anti-phosphotyrosine antibody of immunoprecipitated Src showed that the hormone decreased Src tyrosine phosphorylation state with maximal effects at 5 min. Using a database for protein consensus motifs we found a putative tyrosine phosphorylation site (amino acids 164-170: KTFDTTY) within the primary sequence of the chick VDR. When the myotube VDR was immunoprecipitated it appeared onto SDS-PAGE gels as a single band of 58 kDa recognized by an anti-phosphotyrosine antibody. Prior treatment of cells with (1)alpha,25(OH(2))D(3) significantly increased tyrosine phosphorylation of the VDR (two- to three-fold above basal levels). In agreement with Src being a SH2-domain containing protein involved in recognition of tyrosine-phosphorylated targets, immunoprecipitation with anti-Src antibody under native conditions followed by blotting with anti-VDR antibody, or using the antibodies in inverse order, showed that the VDR co-precipitates with Src, thus indicating the existence of a VDR/Src complex. Stimulation with the cognate VDR ligand significantly increased formation of the complex with respect to basal conditions. These results altogether provide the first evidence to date for 1 alpha,25(OH(2))D(3) activation involving Src association to tyrosine phosphorylated VDR.
Buitrago
C
C
Departamento de Biologia, Bioquímica y Farmacia, Universidad Nacional del Sur, San Juan 670, (8000) Bahia Blanca, Argentina.
Vazquez
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G
De Boland
A R
AR
Boland
R L
RL
D000818:Animals; D002117:Calcitriol / Q000494:pharmacology*; D002642:Chick Embryo; D004789:Enzyme Activation; D018482:Muscle, Skeletal / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D010766:Phosphorylation; D018167:Receptors, Calcitriol / Q000378:metabolism*; D015398:Signal Transduction; D014443:Tyrosine / Q000378:metabolism*; D019061:src-Family Kinases / Q000378:metabolism*
D018167:Receptors, Calcitriol; D014443:Tyrosine; D019061:src-Family Kinases; D002117:Calcitriol