5 research outputs found
Identification of casein kinase 1, casein kinase 2, and cAMP-dependent protein kinase-like activities in Trypanosoma evansi
Identification of Casein Kinase 1, Casein Kinase 2, and cAMPdependent Protein Kinase-like Activities in Trypanosoma evansi
Trypanosoma evansi contains protein kinases capable of phosphorylating
endogenous substrates with apparent molecular masses in the range
between 20 and 205 kDa. The major phosphopolypeptide band, pp55, was
predominantly localized in the particulate fraction. Anti-α and
anti-β tubulin monoclonal antibodies recognized pp55 by Western
blot analyses, suggesting that this band corresponds to phosphorylated
tubulin. Inhibition experiments in the presence of emodin, heparin, and
2,3-bisphosphoglycerate indicated that the parasite tubulin kinase was
a casein kinase 2 (CK2)-like activity. GTP, which can be utilized
instead of ATP by CK2, stimulated rather than inactivated the
phosphorylation of tubulin in the parasite homogenate and particulate
fraction. However, GTP inhibited the cytosolic CK2 responsible for
phosphorylating soluble tubulin and other soluble substrates. Casein
and two selective peptide substrates, P1 (RRKDLHDDEEDEAMSITA) for
casein kinase (CK1) and P2 (RRRADDSDDDDD) for CK2, were recognized as
substrates in T. evansi. While the enzymes present in the soluble
fraction predominantly phosphorylated P1, P2 was preferentially labeled
in the particulate fractions. These results demonstrated the existence
of CK1-like and CK2-like activities primarily located in the parasite
cytosolic and membranous fractions, respectively. Histone II-A and
kemptide (LRRASVA) also behaved as suitable substrates, implying the
existence of other Ser/Thr kinases in T. evansi. Cyclic AMP only
increased the phosphorylation of histone II-A and kemptide in the
cytosol, demonstrating the existence of soluble cAMP-dependent protein
kinase-like activities in T. evansi. However, no endogenous substrates
for this enzyme were identified in this fraction. Further evidences
were obtained by using PKI (6-22), a reported inhibitor of the
catalytic subunit of mammalian cAMP-dependent protein kinases, which
specifically hindered the cAMP-dependent phosphorylation of histone
II-A and kemptide in the parasite soluble fraction. Since the sum of
the values obtained in the parasite cytosolic and particulate fractions
were always higher than the values observed in the total T. evansi
lysate, the kinase activities examined here appeared to be inhibited in
the original extract
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The gene product of a Trypanosoma equiperdum ortholog of the cAMP-dependent protein kinase regulatory subunit is a monomeric protein that is not capable of binding cyclic nucleotides
The full gene sequence encoding for the Trypanosoma equiperdum ortholog of the cAMP-dependent protein kinase (PKA) regulatory (R) subunits was cloned. A poly-His tagged construct was generated [TeqR-like(His)8], and the protein was expressed in bacteria and purified to homogeneity. The size of the purified TeqR-like(His)8 was determined to be ∼57,000 Da by molecular exclusion chromatography indicating that the parasite protein is a monomer. Limited proteolysis with various proteases showed that the T. equiperdum R-like protein possesses a hinge region very susceptible to proteolysis. The recombinant TeqR-like(His)8 did not bind either [3H] cAMP or [3H] cGMP up to concentrations of 0.40 and 0.65 μM, respectively, and neither the parasite protein nor its proteolytically generated carboxy-terminal large fragments were capable of binding to a cAMP-Sepharose affinity column. Bioinformatics analyses predicted that the carboxy-terminal region of the trypanosomal R-like protein appears to fold similarly to the analogous region of all known PKA R subunits. However, the protein amino-terminal portion seems to be unrelated and shows homology with proteins that contained Leu-rich repeats, a folding motif that is particularly appropriate for protein-protein interactions. In addition, the three-dimensional structure of the T. equiperdum protein was modeled using the crystal structure of the bovine PKA RIα subunit as template. Molecular docking experiments predicted critical changes in the environment of the two putative nucleotide binding clefts of the parasite protein, and the resulting binding energy differences support the lack of cyclic nucleotide binding in the trypanosomal R-like protein