JBC, Vol. 250, Issue 7, 2546-2555, Apr, 1975
Phosphorylation of ribosomal proteins in rat cerebral cortex in vitro
C. D. Ashby and S. Roberts
Incubation of cerebral cortical tissue from immature rats in the presence
of [32P]orthophosphate resulted in similar rates of incorporation of
radioactivity into the proteins of free and membrane-bound ribosomes.
Incorporation of label into ribosomal proteins of both species continued
actively for at least 3 hours. Since recovery of membrane-bound ribosomes
from rat cerebral cortex is quite low, further analyses of the radioactive
phosphoproteins were restricted to the free ribosome population. A
significant fraction of the radioactivity which was precipitated with
trichloroacetic acid was not removed by heating in trichloroacetic acid at
90 degrees or extracted with organic solvents and therefore was presumed to
be covalently bound to protein. The radioactive phosphoryl groups present
in the ribosomal proteins were mainly in ester linkages since they were
readily removed by exposure to 1 N NaOH, relatively unaltered by 1N HCl,
and unaffected by hydroxylamine. This conclusion was supported by the
isolation of labeled o-phosphoserine and o-phosphothreonine residues from
hydrolysates of ribosomal proteins. A significant fraction of the labeled
phosphoproteins in the purified ribosomes appeared to be bound tightly to
the ribosome structure since only 40% of the radioactivity could be removed
by extraction of these ribosomes with 1 M KCl. Phosphorylation of proteins
of cerebral monoribosomes was more rapid than the same process in
polyribosomes from the same source. Eight radioactive phosphoprotein bands
could be detected by electrophoresis of proteins obtained from
unfractionated cerebral ribosomes on unidimensional polyacrylamide gels
containing sodium dodecyl sulfate. The protein nature of these materials
was confirmed by pronase digestion. Proteins of subribosomal particles
isolated from the total free ribosomal population were labeled
differentially. When dissociation was carried out in the presence of EDTA,
the small subunit contained four radioactive phosphoprotein bands, whereas
the large subunit contained five. Three of the radioactive phosphoprotein
components of the small subunit were removed when dissociation of cerebral
ribosomes which were previously washed with high salt media was carried out
in the presence of puromycin and high salt. However, only the largest
labeled phosphoprotein band of the large subunit was removed by this
procedure. This component exhibited the same electrophoretic mobility as
one of the radioactive phosphoprotein bands which was removed from the
small subunit by high salt treatment..
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