Identification and specific localization of tyrosine-phosphorylated proteins in Trypanosoma brucei

Abstract

Phosphorylation on tyrosine residues is a key signal transduction mechanism known to regulate intercellular and intracellular communication in multicellular organisms. Despite the lack of conventional tyrosine kinases in the genome of the single cell organism Trypanosoma brucei, phosphorylation on trypanosomal protein tyrosine residues has been reported for this parasite. However, the identities of most of the tyrosine-phosphorylated proteins and their precise site(s) of phosphorylation were unknown. Here, we have applied a phosphotyrosine-specific proteomics approach to identify 34 phosphotyrosine-containing proteins from whole-cell extracts of procyclic form T. brucei. A significant proportion of the phosphotyrosine-containing proteins identified in this study were protein kinases of the CMGC kinase group as well as some proteins of unknown function and proteins involved in energy metabolism, protein synthesis, and RNA metabolism. Interestingly, immunofluorescence microscopy using anti-phosphotyrosine antibodies suggests that there is a concentration of tyrosine-phosphorylated proteins associated with cytoskeletal structures (basal body and flagellum) and in the nucleolus of the parasite. This localization of tyrosine-phosphorylated proteins supports the idea that the function of signaling molecules is controlled by their precise location in T. brucei, a principle well known from higher eukaryotes.

FIG. 1.

Detection of T. brucei tyrosine-phosphorylated proteins by immunoblotting. Aliquots of total cell lysates (T) and TX-100-soluble (S) and insoluble (P) fractions from 6 × 10⁶ procyclic form trypanosome equivalents were subjected to SDS-polyacrylamide gel electrophoresis and Western blotting with 4G10 antibody before (Control, lanes 1 to 3) and after alkaline phosphatase (alk. phosphatase, lanes 4 to 6) or SHP-1 phosphotyrosine-specific phosphatase (SHP-1, lanes 7 to 9) treatment of the blot. Following the 4G10 Western blotting, the membranes were stripped and probed with anti-tubulin antibodies as a loading control (lower panels). α-pTyr, anti-phosphotyrosine.

FIG. 2.

T. brucei cells are sensitive to treatment with hydrogen peroxide. Procyclic trypanosomes were treated with 200 μM, 400 μM, and 800 μM hydrogen peroxide for 10 min, 20 min, and 30 min before cell lysis. An aliquot of 15 μg protein was loaded in each lane. Lane 1, protein lysate of untreated cells. Bands that show a clear response to the hydrogen peroxide treatment are indicated with an asterisk. α-pTyr, anti-phosphotyrosine.

FIG. 3.

Mass spectrometric analysis of T. brucei phosphotyrosine-containing peptides. Shown is the fragmentation spectrum of the diphosphorylated peptide GVGVNVpTSpYVVTR (p indicates phosphorylated residue) of a putative TbMAPK (GeneDB accession no. Tb927.6.1780) measured on an LTQ-Orbitrap mass spectrometer. Phosphorylation at the threonine and tyrosine residues of the TSY motif could be deduced due to the neutral loss of phosphoric acid starting from the y₇ and b₇ ions (−P) and the observed mass increment of 243 Da (+P) between the y₄ and y₅ ions, respectively.

FIG. 4.

Indirect immunofluorescence using anti-phosphotyrosine antibodies. Whole-cell procyclic trypanosomes were fixed in 4% PFA and stained with 4G10 (A), PY-20 (B), and PY-100 (C). (D) A no-primary-antibody control was included and demonstrated that the staining was a consequence of the primary anti-phosphotyrosine antibodies. Tyrosine-phosphorylated proteins (in green) localized to punctate structures in the posterior of the cells (white arrows) and were also found along the length of the flagellum (yellow arrows) and in the nucleus (white arrowheads). (E and F) Control HeLa cells labeled with 4G10 showed concentrated anti-phosphotyrosine staining (in red) at focal adhesion regions. DNA (blue) is visualized by 4′-6-diamidino-2-phenylindole (DAPI) staining. Anti-α-tubulin is shown in green. p-Tyr, tyrosine-phosphorylated proteins. White bar corresponds to a length of 5 μm.

FIG. 5.

Indirect immunofluorescence analysis of cytoskeletal preparations using anti-phosphotyrosine antibodies. Procyclic form cytoskeletons were fixed in 4% PFA and stained with 4G10 (A), PY-20 (B), and PY-100 (C). (D) A no-primary-antibody control was included and demonstrated that the staining was a consequence of the primary anti-phosphotyrosine antibodies. The staining pattern suggested labeling of the basal bodies in the posterior of the cell and was observed along the length of the flagellum (in green). DNA (blue) is visualized by 4′-6-diamidino-2-phenylindole (DAPI) staining. White bar corresponds to a length of 5 μm.

FIG. 6.

Colocalization studies of phosphotyrosine staining using the anti-basal body marker BBA4. Indirect immunofluorescence of procyclic form T. brucei cytoskeletons colabeled with the anti-phosphotyrosine antibody 4G10 (A) and BBA4 (B), a specific marker for the proximal pole of both basal and probasal structures. Images shown in panels A and B are shown merged in panel C. Differential interference contrast (DIC) images of T. brucei flagella were taken (D) and merged with immunofluorescence images labeled with the anti-phosphotyrosine 4G10 antibody (E). White arrows, labeling of the basal body. Colabeling structures that were observed are enlarged and surrounded by a white box. p-Tyr, tyrosine-phosphorylated proteins. White bar corresponds to a length of 5 μm.

FIG. 7.

Phosphotyrosine-containing proteins are associated with the flagellum axoneme. Cytoskeleton preparations of procyclic form T. brucei (A) were colabeled with the anti-phosphotyrosine 4G10 antibody (B) and the anti-axonemal Rib72 antibody (C). The overlap of both staining patterns in most of the areas (D) strongly suggested that the anti-phosphotyrosine signal is axonemal. The area of the phosphotyrosine signal that did not colocalize with the Rib72 staining is indicated with arrows (D). White bar corresponds to a length of 5 μm.