INVESTIGADORES
REY Osvaldo
artículos
Título:
Activation loop Ser744 and Ser748 in protein kinase D are transphosphorylated in vivo.
Autor/es:
WALDRON RT, REY O, IGLESIAS T, TUGAL T, CANTRELL D, ROZENGURT E.
Revista:
JOURNAL OF BIOLOGICAL CHEMISTRY
Editorial:
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
Referencias:
Año: 2001 p. 32606 - 32615
ISSN:
0021-9258
Resumen:
The importance of activation loop phosphorylation in the regulation of
protein kinase D (PKD/protein kinase C (PKC) mu) activity has become
controversial. In order to clarify the mechanism(s) of PKD activation,
we developed a novel phosphospecific antibody recognizing
phosphorylated Ser(748) in PKD (pS748). Western blot analysis with the
pS748 antibody, carried out with a variety of PKD forms and in a
variety of cell types including full-length PKD transfected in COS-7
and HEK 293 cells, a green fluorescent protein-PKD fusion protein
transfected in either Swiss 3T3 fibroblasts or Madin-Darby canine
kidney epithelial cells, and endogenous PKD expressed in A20
lymphocytes and Rat-1 fibroblasts, indicated that Ser(748)
phosphorylation was absent from unstimulated cells. In contrast,
dramatic increases in Ser(748) phosphorylation were induced by phorbol
esters, bombesin, or cross-linking of B lymphocyte antigen receptors or
by cotransfection with active PKCepsilon or PKCeta. Western analysis
using a second phosphospecific antibody, which primarily recognizes PKD
phosphorylated at Ser(744), revealed that Ser(744) phosphorylation
accompanies Ser(748) phosphorylation during PKD activation in vivo.
Ser(744)/Ser(748) phosphorylation requires PKC but not PKD activity,
indicative of transphosphorylation. Our results provide new
experimental evidence indicating that activation loop phosphorylation
at Ser(744) and Ser(748) occurs during PKD activation in vivo and
support the notion of a PKC-PKD phosphorylation cascade.