M. Parreno et al., E1A modulates phosphorylation of p130 and p107 by differentially regulating the activity of G1/S cyclin/CDK complexes, ONCOGENE, 20(35), 2001, pp. 4793-4806
We have previously shown that the adenoviral 12S E1A protein modulates the
phosphorylation status of p130 and p107 without apparent changes in the cel
l cycle dependent phosphorylation of the retinoblastoma protein. Here we re
port on the mechanisms by which E1A modifies differentially the phosphoryla
tion status of pocket proteins. In human U-2 OS osteosarcoma cells transien
tly expressing E1A, ectopic expression of D-type cyclins alone or combined,
but not cyclins E and/or A, fully rescues EIA-mediated block in hyperphosp
horylation of p130 to form 3. However, cyclins E and A, individually or tog
ether, induce hyperphosphorylation of p130 to species with intermediate mob
ility. Phosphopeptide maps indicate that E1A inhibits phosphorylation of si
tes phosphorylatable by CDKs. One of these sites is Ser-1044. The effects o
f blocking the activities of endogenous and exogenous cyclins with p16 and
dominant negative CDK2 in E1A expressing cells further indicate that p130 i
s phosphorylated by both D-type cyclin and cyclin E/CDK complexes and that
E1A modulates the activity of these G1/S CDKs by independent mechanisms. St
able expression of E1A in MC3T3-E1 cells leads to downregulation of D-type
cyclins, and upregulation of cyclins E and A. This is accompanied by increa
sed CDK2 kinase activity. Downregulation of D-type cyclins in these cells c
orrelates with a block on both p130 hyperphosphorylation to form 3 and hype
rphosphorylation of p107. This is rescued by D-type cyclins but not by cycl
in E. In addition, we show that the upregulation of cyclins E and A is at l
east partially dependent on an intact pocket protein/E2F pathway, but downr
egulation of D-type cyclins is not. Moreover, we provide evidence that whil
e the lack of a functional pRB pathway also results in a block on hyperphos
phorylation of p130 to form 3, this is not sufficient to induce constitutiv
e expression of p130 form 2b.