Ad. Xu et al., Serine phosphorylation of the sarcoplasmic reticulum Ca2+-ATPase in the intact beating rabbit heart, BIOC BIOP R, 264(1), 1999, pp. 241-246
Citations number
37
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Recent studies have demonstrated that Ca2+/calmodulin-dependent protein kin
ase phosphorylates the Ca2+-pumping ATPase of cardiac sarcoplasmic reticulu
m (SR) in vitro. Also, evidence from in vitro studies suggested that this p
hosphorylation, occurring at Ser(38), results in stimulation of Ca2+ transp
ort. In the present study, we investigated whether serine phosphorylation o
f the SR Ca2+-ATPase occurs in the intact functioning heart. Hearts removed
from anesthetized rabbits were subjected to retrograde aortic perfusion of
the coronary arteries with oxygenated mammalian Ringer solution containing
P-32(i) and contractions were monitored by recording systolic left ventric
ular pressure development. Following 45-50 min of P-32 perfusion, the heart
s were freeze-clamped, SR isolated, and analyzed for protein phosphorylatio
n. SDS-polyacrylamide gel electrophoresis and autoradiography showed phosph
orylation of several peptides including the Ca2+-ATPase and Ca2+ release ch
annel (ryanodine receptor). The identity of Ca2+-ATPase as a phosphorylated
substrate was confirmed by Western immunoblotting as well as immunoprecipi
tation using a cardiac SR Ca2+-ATPase-specific monoclonal antibody. The Ca2
+-ATPase showed immunoreactivity with a phosphoserine monoclonal antibody i
ndicating that the in situ phosphorylation occurred at the serine residue.
Quantification of Ca2+-ATPase phosphorylation in situ yielded a value of 20
8 +/- 12 pmol P-32/mg SR protein which corresponded to the phosphorylation
of similar to 20% of the Ca2+ pump units in the SR membrane. Since this pho
sphorylation occurred under basal conditions (i.e., in the absence of any i
notropic intervention), a considerable steady-state pool of serine-phosphor
ylated Ca2+-ATPase likely exists in the normally beating heart. These findi
ngs demonstrate that serine phosphorylation of the Ca2+-ATPase is a physiol
ogical event which may be important in the regulation of SR function. (C) 1
999 Academic Press.