trans

274 INTRACELLULAR SIGNALING
PfPPJ is not sensitive to okadaic acid, microcystin-LR,
and the PP1-specific mammalian
heat-stable inhibitor-2. These observations
suggest that PfPPJ does not belong to either
the PP2A or PP1 class. Additionally, PfPPJ does
not require calmodulin or Ca 2 for its catalytic
activity and therefore does not belong to the
PP2B class. PfPPJ is developmentally regulated
and shows peak expression predominantly in
the schizont stage.
Another phosphatase from P. falciparum is
an atypical PP2C homolog. The predicted
amino acid sequence of PfPP2C can be
divided into two functional halves, PfPP2C-1
and PfPP2C-2. PfPP2C-1, PfPP2C-2, or the fulllength
PfPP2C complement S. pombe PP2C
temperature-sensitive mutants. Recombinant
protein derived from each half of PP2C is
catalytically active. PfPP2C selectively dephosphorylates
PKC-mediated phosphorylation of
translation elongation factor-1.
Protein phosphatases from kinetoplastids
Initial evidence for an important role of
protein phosphatases in cell growth and differentiation
of trypanosomatids came from the
treatment of cells with okadaic acid. In
T. brucei, okadaic acid treatment uncouples
the nuclear and organellar division resulting in
cells with multiple nuclei and a single kinetoplast.
In T. cruzi, calyculin A-treated cells can
undergo mitosis in the absence of cytokinesis.
However, unlike in T. brucei, T. cruzi epimastigotes
are relatively insensitive to okadaic acid.
Two PP1 homologs, TcPP1 and TcPP1 have
been cloned. Interestingly, TcPP1 is significantly
more sensitive to calyculin A than
okadaic acid (IC 50 of 2 nM vs. 100 nM). A PP5-
type T. brucei protein phosphatase (TbPP5)
also has been characterized. It contains PP5
signature tetratricopeptide repeats at the
amino terminus and the phosphatase catalytic
domain at the carboxy terminus. The 52 kDa
TbPP5 is expressed in both bloodstream and
procyclic forms. TbPP5 is found predominantly
in the cytoplasm although some nuclear localization
has also been observed. The recombinant
TbPP5 is catalytically active and shows
modest stimulation by arachidonic acid. In
Leishmania chagasi PP1 and PP2C-like activities
have been detected, the PP2C (LcPP2C)
gene has been cloned, and the recombinant
protein is enzymatically active.
In summary, reversible phosphorylation
mediated by an array of kinases and phosphatases
is expected to play a key role in various
cellular processes of protozoan parasites.
Although a variety of kinases and phosphatases
have been isolated from different
species based on homology, their exact physiological
roles are not clear. Ascertaining the role
of these proteins will be a major challenge of
the post-genome-project era. Many of these
proteins may have evolved unusual functions
as a means of controlling complex life-cycle
events. These atypical characteristics will not
be evident from primary amino acid sequences.
Novel structure and properties of parasite
kinases and phosphatases may be exploited
in the design of new generation anti-parasitic
therapeutics with a high degree of selectivity.
CONCLUSIONS
Overall it is evident that protozoan parasites
contain a complex web of signaling components.
Evolutionarily conserved enzymes
involved in Ca 2 sensing, cyclic nucleotide
metabolism, and phosphoryl transfer reactions
have been identified. While many of these
enzymes share homology with host proteins,
others appear to be unique in structure or function.
Inhibitor studies and the more limited
gene knockout studies hint at the essential
BIOCHEMISTRY AND CELL BIOLOGY: PROTOZOA