Protein kinase A catalytic subunit interacts and phosphorylates members of trans-sialidase super-family in Trypanosoma cruzi.
Protein kinase A (PKA) has been suggested as a regulator of stage differentiation in Trypanosoma cruzi. Using a yeast two-hybrid system we have begun to characterize the downstream substrates of T. cruzi PKA. We identified several members of the trans-sialidase super family by this approach. Immunoprecitation demonstrated that a TcPKAc monoclonal antibody was able to pull-down proteins recognized by trans-sialidase antibodies as well as a SA85-1.1 antibody and vice versa. An in vitro phosphorylation assay demonstrated that PKA phosphorylated the recombinant protein of an active trans-sialidase. In addition, a phospho-(Ser/Thr) PKA substrate antibody detected bands on immunoblot analysis of trans-sialidase antibody precipitated proteins from parasite lysate and the media of L(6)E(9) myoblasts infected with trypomastigotes as well as from a SA85-1.1 antibody precipitated proteins from parasite lysate. Immunofluorescence analysis suggested that some TcPKAc localizes to the plasma membrane surface of trypomastigotes. The identified trans-sialidases have PKA consensus phosphorylation sites located near the endoplasmic reticulum retention motif in the N-terminal. These data support that PKA phosphorylates trans-sialidase super family members in vivo.
Bao
Yi
Y
Department of Pathology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
Weiss
Louis M
LM
Ma
Yan Fen
YF
Kahn
Stuart
S
Huang
Huan
H
D000818:Animals; D020134:Catalytic Domain; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism*; D054884:Host-Pathogen Interactions; D015151:Immunoblotting; D047468:Immunoprecipitation; D008856:Microscopy, Fluorescence; D009439:Neuraminidase / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D015800:Protozoan Proteins / Q000378:metabolism*; D051381:Rats; D014349:Trypanosoma cruzi / Q000472:pathogenicity*; D020798:Two-Hybrid System Techniques
D006023:Glycoproteins; D015800:Protozoan Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; C069715:trans-sialidase; D009439:Neuraminidase
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