trans

REVERSIBLE PROTEIN PHOSPHORYLATION 269
presence of truncated isoforms of 40 and
80 kDa that are tyrosine phosphorylated. The
40 kDa isoform could be a proteolytic degradation
product, and the 80 kDa form could
be the full-length protein that migrates anomalously
on gels. PfMAP-1 expression is detected
in both asexual and sexual stages. PfMAP-2
appears to be a novel protein, although it
contains significant homology to MAPKs.
Recombinant PfMAP-2 exhibits typical MAPK
properties such as autophosphorylation, phosphorylation
of myelin basic proteins, and the
reduction in activity following treatment with
MAPK-specific phosphatases. However, the
signature TXY activation sequence is substituted
in PfMAP-2 with TSH. Replacement of
the threonine in TSH with alanine abolishes
autophosphorylation of PfMAP-2, suggesting
a role for this residue in catalysis. PfMAP-2
expression is specific for gametocytes.
In a search for the MAPK-activating kinases
MAPKK or MEK, the P. falciparum genomesequence
database has been searched using
the conserved activation site sequences found
in MEK1 and MEK2. A 996-amino acid protein
with a long carboxy terminus extension and
many conserved residues of the MEK family
was identified. The putative Pfnek-1 catalytic
domain however, shows highest homology to
NIMA (never in mitosis)-like kinases, a family
of cyclin-independent kinases required for
progression into mitosis. Pfnek-1 phosphorylates
PfMAP-2, but not PfMAP-1. Pfnek-1 also
stimulates the ability of PfMAP-2 to phosphorylate
myelin basic protein in vitro. Both
PfMAP-2 and Pfnek-1 are expressed in gametocytes,
suggesting that Pfnek-1 may be
involved in PfMAP-2 activation during sexual
stage differentiation.
MAP kinase pathways in kinetoplastids
More direct proof of a role for MAPKs in parasite
differentiation comes from Leishmania
mexicana. Deletion of the tandemly repeated
secreted acid phosphatase (SAP) gene locus
(lmsap1/2) curtails the transformation of
promastigotes into amastigotes within
macrophages. Analysis of the lmsap1/2 locus
revealed that a MAPK homolog (termed
LMPK) is encoded by the intergenic region
between the sap genes. LMPK contains signature
MAPK sequences. Interestingly, the lmpk
gene is sufficient to complement the lmsap1/2
null mutation, as evidenced by progression
of infection and appearance of amastigotes
in transfected cells. Although the LMPK protein
is expressed in both promastigotes and
amastigotes, LMPK activity is found only in
amastigotes, suggesting that appropriate activators,
such as MAPKKs, are present only in
amastigotes.
In T. brucei, a MAPK homolog, termed
KFR1, with a strong homology to yeast MAPK
homologs KSS1/FUS3, has been cloned. The
KFR1 gene is presumed to be essential to
T. brucei procyclic forms, since viable cells
could not be obtained when the gene was
disrupted at both alleles. Recombinant KFR1
can utilize myelin basic protein as a substrate
in vitro, in common with other MAPKs.
Phosphorylation of KFR1 is important for
activity, and dephosphorylation by MAPK
(ERK1 and ERK2)-specific dual phosphatase
abolishes activity. The specific activity of KFR1
varies during the life cycle, and is higher in
bloodstream forms than procyclic forms.
When an extra copy of KFR1 is introduced
into procyclic forms, an increased abundance
of KFR1 transcripts is observed. However, the
amount of KFR1 protein remains constant,
indicating that the level of KFR1 is translationally
controlled. Therefore, trypanosomes exert
two levels of control over KFR1: regulating the
amount of KFR1 by translational control, and
the activity of KFR1 by phosphorylation. Serum
starvation of bloodstream T. brucei causes
BIOCHEMISTRY AND CELL BIOLOGY: PROTOZOA