Bariloche protein symposium argentine society for biochemistry and molecular biology

56
BIOCELL, 27 (Suppl. I), 2003
TS-C2.
CDK4/6 INHIBITOR p19INK4d INCREASES THE DNA
REPAIR ABILITY IN FIBROBLAST
Cánepa, Eduardo; Julio, Miguel; Ceruti, Julieta; Carcagno, Abel;
Guberman, Alejandra and Scassa, María.
Laboratorio de Biología Molecular, Depto. Química Biológica,
FCEN, UBA, Buenos Aires, Argentina. E-mail:
[e-mail]
The four proteins of the INK4 family posses a similar structure
and bind to CDK4/6 with similar affinity driving to a G1 cell
cycle arrest, but they are reported to have different biological roles.
The aim of this work was to evaluate if p19
INK4d
 induces a second
photoprotective response after DNA damage. Northern blot analysis
show that p19 is up-regulated after UV irradiation in BHK cells.
Levels of p19
INK4d
 mRNA peaked 12 h after UV treatment. This
induction resulted dose dependent and started at 5 mJ/cm
2
. The
apparent p19
 
mRNA half life was 3 h in irradiated and non
irradiated cells. The same behavior was observed when the protein
half-life was estimated in 2,5 h by pull-chase assays with
 35
S-met.
Nuclear run-on experiments revealed an appreciable increase in
the rate of p19
 
mRNA synthesis in UV treated cells in comparison
with untreated ones. We measured DNA repair capacity by host
cell reactivation assay. The overexpression of p19 resulted in a
80% increased repair of UV-induced DNA damage while the
antisense version drove a diminished expression of DNA tested.
Nevertheless, mimosine (an inhibitor of G1/S phase progression)
or overexpression of p16
INK4a
 displayed a minor effect on the rate
of repair as assessed by HCR, suggesting that p19
INK4d 
exerts its
action independently from cell cycle arrest. Regardless of p19
INK4d
precise mechanism the present data demonstrate that this protein
is not only associated with cell cycle arrest but also with
enhancement of DNA repair.
TS-C3.
STUDIES ON ACTIVATION AND PHOSPHORYLATION
OF PROTEIN KINASE A DURING THE TRANSITION
FROM  RESPIRATORY TO FERMENTATIVE
METABOLISM IN SACCHAROMYCES CEREVISIAE
P. Portela and  S. Moreno.
Departamento de Química Biológica, Facultad de Ciencias
Exactas y Naturales, Universidad  de  Buenos Aires. E-mail:
[e-mail]
Protein kinase A (PKA) activity was measured in vivo after addition
of glucose to cells growing on a non-fermentable carbon source, a
physiological condition known to produce a transient peak of
cAMP. PKA activity followed in time the peak of cAMP. The PKA
activity measured in presence of 10 
µM of cAMP, also showed a
peak of activity. The level of cAMP intracellularly bound to
regulatory subunit followed the course of PKA activation. Western
blots for regulatory and catalytic subunits and their corresponding
activities, assayed in partially purified samples, showed constant
levels of both subunits. These results suggest a different state of
activability of PKA within the cells, as a consequence of binding
of cAMP along the transient rise of cAMP triggered by glucose
addition. The relative proportion of phosphorylated species of the
catalytic subunit (TPK1), changed during the transition from non-
fermentable to fermentable carbon source, in a peak of cAMP and
PKA activity-dependent manner. The phosphorylation state of
TPK1 was found carbon source dependent: glycerol-grown cells
have less phosphorylated isoforms than glucose-grown cells.
Preliminary kinetic analysis of crude extracts, carryng
phosphorylated isoforms before and  after phosphatase treatment
showed that the phosphorylation of TPK1 decreases the Km for
the kemptide.
TS-C4.
TYROSINE PHOSPHATASES ACT ON
STEROIDOGENESIS THROUGH THE ACTIVATION OF
AA RELEASE
F. Cornejo Maciel, F. Cano, E.J. Podestá.
Dept. Biochemistry, School of Medicine, UBA. E-mail:
[e-mail]
We have previously demonstrated a hormone-dependent activation
of protein tyrosine phosphatases (PTPs) in adrenocortical and
Leydig cells, involved in the cAMP/PKA dependent induction of
StAR protein (Steroidogenic Acute Regulatory protein) and
activation of steroidogenesis. We also probed that arachidonic acid
(AA) release is another key step in the action of StAR protein.
Therefore, we questioned whether PTPs and AA release are linked
in the regulation of the whole process. Given that AA levels are
regulated by the concerted action of an acyl-CoA synthetase (ACS4)
and an acyl-CoA thioesterase (ARTISt), we tested the effect of
PTP inhibitors on ACS4 expression. We used two steroidogenic
cell lines, MA-10 and Y1, stimulated with 8Br-cAMP in the
presence and absence of benzylphosphonic acid (BPA, a PTP
inhibitor). ACS4 expression, increased after cAMP treatment, was
abolished by PTP inhibition. The involvement of PTPs in AA
release was also tested using exogenous AA to bypass PTP
inhibition, evaluating the effect on progesterone (P4) biosynthesis
by MA-10 Leydig cells. P4 production (ng P4/ml) stimulated by 1
mM 8Br-cAMP was inhibited by 0.2 mM BPA (7.6 
± 0.8 vs 2.3 ±
0.4), as already described. AA itself (0.3 mM) has a stimulatory
effect (5.3 
± 0.6) and reverses BPA inhibition (6.5 ± 0,7). These
results, together with the fact that PTP inhibitors block hormone
induction of ACS4, strongly suggest that the stimulation of
steroidogenesis involves AA release downstream of PKA and PTPs
activation.
TS-C5.
EXPRESSION OF PROTEIN PHOSPHATASES FROM THE
PP2A FAMILY IN POTATO PLANTS
Vozza N, Raíces M, Téllez-Iñón MT.
INGEBI-CONICET y FCEyN-UBA. Vuelta de Obligado 2490 2
do
piso, (1428) Buenos Aires, Argentina. E-mail: [e-mail]
Reversible phosphorylation of proteins is known to be important
in the control of plant metabolism, but relatively few proteins
whose activities are controlled by phosphorylation have so far been
identified. In a previows work, two cDNAs encoding putative
catalytic subunits of serine-threonine protein phosphatases from
the protein phosphatase 2A family (StPP2A1 and StPP2A2) were
identified in Solanum tuberosum using a cDNA library from potato
tuberizing stolons. Both deduced aminoacid sequences share 80%
identity and a high degree of similarity with other plant PP2As. In
this work, the expression of StPP2A1 and StPP2A2 was analyzed
using gene specific semiquantitative PCRs. StPP2A1 was
expressed in all the tissues analyzed but presented higher levels
of expression in induced stolons. On the other hand, StPP2A2
was not expressed in leaves or early stolons, it was present in
flowers and tubers and it was highly expressed in stolons induced
to tuberize.
Potato tuberization is a complex process that results in the
differentiation of a stolon into a tuber. The fact that both StPP2A1
and StPP2A2 are upregulated during tuber morphogenesis suggests
that these genes could play a role during this developmental
process.

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