Worm Breeder's Gazette 14(4): 28 (October 1, 1996)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Detection of Ser/Thr protein phosphatase activities in C.elegans

Tamas Zeke, Pal Gergely, Viktor Dombradi

Department of Medical Chemistry, University Medical School of Debrecen, H-4026 Debrecen, Bem ter 18/B, Hungary, e-mail: dombradi@jaguar.dote.hu

        Protein phosphatases (PP) are significant regulatory enzymes
of  all eukaryotes. They must be present in C.elegans as witnessed
by  DNA-deduced   PP-like  amino  acid sequences unraveledin the
framework  of  the  genom  project.  About half of  them belong  to
Ser/Thr  PP,  while the rest is  Tyr  PP.  For the assay of  the main
Ser/Thr   PP classes, namely PP1, PP2A, PP2B  and  PP2C  Cohen
and   coworkers  suggested  a  straightforward and relatively simple
procedure  (1) that was adopted for the analysis of the worm.
        Rabbit muscle phosphorylase a phosphorylated by phospho-
rylase kinase, rabbit muscle inhibitor-1 and casein from bovine milk
phosphorylated by bovine heart cAMP-dependent  protein  kinase
were  used  as  substrates.  C.elegans  Var.  Bristol  (N2)  wild type
strain were cultured,  harvested,  rinsed  with the  extraction  buffer,
frozen in liquid nitrogen and homogenized in four volumes of the ice
cold extraction buffer  (50 mM Tris-HCl  pH=7.4;  0.1 mM EDTA;
10 mM DTT; 0.5% Triton X-100; 2mM PMSF; 5mM benzamidine
and 1 mM o-phenantroline).The homogenates were cleared by cent-
rifugation at 14000  g,  4 oC  for  10  min and than were kept  frozen
in liquid nitrogen till the assays.
    Rabbit skeletal muscle inhibitor-2 (unphosphorylated) was  used
as a specific inhibitor ofPP1 with 32P-phosphorylase a substrate in
the presence ofEDTA,a metal ion chelator(Fig.1).The titration curve
reveals that about 75%of the total activity was supplied by PP1 and
5 nM of inhibitor-2  caused  about 50%  inhibition.  Okadaic  acid, a
potent  marine  toxin and tumor promoter, was used to separate PP1
and PP2A activities with 32P-phosphorylase a  substrate in the pre-
sence of  EDTA (Fig.2).  The titration curve with  okadaic acid is bi-
phasic, in the first step about 30% of  the total  activity  is inhibited
with an IC50 of ~0.04 nM and the rest of activity was inhibited in a
second  step  with  an  IC50 of  ~70  nM.  Complete  inhibition was
achieved by 1 micromol/liter okadaic acid concentration. It is known
that  PP2A is more sensitive to okadaic acid than PP1, thus the first
step of the titration can be explained by the presence of PP2A while
the second step can be attributed to PP1 inhibition.
           For the detection of PP2B we used 32P-inhibitor-1  substrate
and  Ca-calmodulin as an activator. Due to the interference of  prote-
ases the 32Pi liberated in the assays was estimated after  ammonium
molybdate extraction(2). About 50 nmol substrate was converted in
1 min  by the extract. ~70% of the  activity was inhibited by  4 mM
EDTA or 1.5 mM trifluoperazine and ~30% was inhibited by 2 nM
okadaic acid  (not shown).  Thus 70%  of  the total activity was due
to PP2B and 30% to PP2A.
          PP2C was assayed with 32P-casein substrate in the presence
of Mg2+-activator. ~10%of the activity was inhibited by inhibitor-2
(PP1)  ~40%  of  the  activity  was inhibited  by  2 nM okadaic acid
(PP2A) and ~50% was stimulated by Mg2+ (PP2C).  Half  maximal
activation  was  achieved  at  2 mM  Mg2+  concentration,  maximal
activation  was measured at 20 mM (Fig.3.).
            Acknowledgements.This work was supported by the grants
OTKA 6005 and 12840.

(1)   Cohen, P., Klump, S., Schelling, D.L. (1989) FEBS Lett. 250,
(2)   Shenolikar, S., Ingebritsen, T.S. (1984) Meth. Enyzmol. 107,

Figure 1.
Inhibition  of  phosphorylase
phosphatase activity by rabbit
muscle  inhibitor-2  in a crude
C.elegans extract.The activity
measured in the  absence  of
inhibitor-2  was 0.8 mU/mg,
and was taken as 100%.

Figure 2.
Inhibition  of   phosphorylase
phosphatase activity by okadaic
acid  in a crude C.elegans  extract.
100% is the same as in Fig.1.

Figure 3.
Stimulation of casein phosphatase
activity by magnesium ions in a
crude  C.elegans  extract.   The
maximal PP2C activity was 0.29
mU/mg and was taken as 100%.