Diacylglycerol-stimulated endocytosis of transferrin in trypanosomatids is dependent on tyrosine kinase activity

Abstract

Small molecule regulation of cell function is an understudied area of trypanosomatid biology. In Trypanosoma brucei diacylglycerol (DAG) stimulates endocytosis of transferrin (Tf). However, it is not known whether other trypanosomatidae respond similarly to the lipid. Further, the biochemical pathways involved in DAG signaling to the endocytic system in T. brucei are unknown, as the parasite genome does not encode canonical DAG receptors (e.g. C1-domains). We established that DAG stimulates endocytosis of Tf in Leishmania major, and we evaluated possible effector enzymes in the pathway with multiple approaches. First, a heterologously expressed glycosylphosphatidylinositol phospholipase C (GPI-PLC) activated endocytosis of Tf 300% in L. major. Second, exogenous phorbol ester and DAGs promoted Tf endocytosis in L. major. In search of possible effectors of DAG signaling, we discovered a novel C1-like domain (i.e. C1_5) in trypanosomatids, and we identified protein Tyr kinases (PTKs) linked with C1_5 domains in T. brucei, T. cruzi, and L. major. Consequently, we hypothesized that trypanosome PTKs might be effector enzymes for DAG signaling. General uptake of Tf was reduced by inhibitors of either Ser/Thr or Tyr kinases. However, DAG-stimulated endocytosis of Tf was blocked only by an inhibitor of PTKs, in both T. brucei and L. major. We conclude that (i) DAG activates Tf endocytosis in L. major, and that (ii) PTKs are effectors of DAG-stimulated endocytosis of Tf in trypanosomatids. DAG-stimulated endocytosis of Tf may be a T. brucei adaptation to compete effectively with host cells for vertebrate Tf in blood, since DAG does not enhance endocytosis of Tf in human cells.

Figure 1. Heterologously expressed GPI-PLCp activates endocytosis of Tf in Leishmania major.

( A ) Leishmania major CC1 promastigotes expressing GPI-PLC (pUTK-GPIPLC) or vector alone (pUTK) were cultured in a medium containing G418 (50 µg/ml). Cells (1×10⁶/ml) were incubated with transferrin-Alexa Fluor 594 (25 µg/ml) at 27°C, and at specified time intervals cell-associated fluorescence was measured. Relative Fluorescence Units was plotted after subtracting background fluorescence of an equivalent number of control cells. Mean±standard deviation of triplicate determinations are plotted. ( B ) Promastigote L. major (pUTK/L. major and pUTK-GPIPLC/L. major) were seeded at a density of 5×10⁵ cells/ml in medium containing G418 (50 µg/ml), and every 24 h cells were counted microscopically. Mean±standard deviation of duplicate measurements is plotted.

Figure 2. Endosome or glycosome-directed GPI-PLC stimulate endocytosis.

Leishmania harboring pUTK-GPIPLCp, pUTK-GPIPLC-C269S/C273S, pUTK-GPIPLC-C269S/C270S/C273S, or pUTK-GPIPLC-Q81L were cultured in medium containing 50 µg/ml G418 and transferrin endocytosis was measured as described in Fig. 1. Representative data from three experiments is shown. (Intracellular location of GPI-PLCp and its Cys mutants has been described , .)

Figure 3. Enzyme activity is important for GPI-PLCp stimulation of Tf Endocytosis in L. major.

L. major pUTK/GPIPLC-Q81L and pUTK-GPIPLC were cultured in 50 µg/ml G418 and allowed to endocytose transferrin-Alexa Fluor 594 at 27°C for indicated time intervals. Cell-associated transferrin is plotted as relative fluorescence units.

Figure 4. Phorbol ester and diacylglycerols activate endocytosis of Tf in L. major.

(A) L major harboring pUTK or pUTK-GPIPLC (1×10⁶/ml) were incubated at 27°C with PMA or 4α-PMA (500 nM; final concentration) for 15 min, or (B) oleoyl-acetyl-sn-glycerol (OAG) or dimyristoyl glycerol (DMG) (250 nM; final concentration) for 30 min. Endocytosis of Tf was measured as described in the legend for Figure 1. Data presented are means (with standard deviations) of triplicate determinations.

Figure 5. A Ser/Thr kinase Inhibitor does not block DAG-activated endocytosis of Tf in T. brucei or L. major.

Bloodstream T. brucei (5×10⁶ cells) ( A ) or L. major promastigotes (1×10⁶ cells) ( C ) were incubated with DMSO (vehicle) or different amounts of Ro32-0432 for 10 min at 37°C (for T. brucei) or 27°C (for L. major). Subsequently, endocytosis of Tf was measured as described earlier. T. brucei ( B ) or L. major ( D ) was incubated in medium containing Ro32-0432 (500 nM) for 10 min (i.e., Stage I). Cells were then exposed to PMA (500 nM) (Stage II) for another 10 min, and endocytosis of Tf was measured. A representative experiment is presented. Data plotted are means (with standard deviations) of triplicate determinations.

Figure 6. Alignment of C1_5 domains in T. brucei.

( A ) C1-like domains of T. brucei proteins (Table 1) are aligned (Clustal W) with LaserGene (DNAStar). A consensus sequence is presented above the alignment. ( B ) Alignment of C1_5 domains in protein Tyr kinases from T. brucei, T. cruzi and L. major (see text for details).

Figure 7. A PTK inhibitor blocks phorbol ester-stimulated endocytosis of Tf in T. brucei and L. major.

( A ) T. brucei (5×10⁶) were incubated with varying concentrations of Tyrphostin A47 for 10 min. Parasites were rinsed, and endocytosis of Tf measured (see legend to Fig. 1 for protocol). ( B ) T. brucei (5×10⁶ cells) was incubated in medium containing Tyrphostin A47 (TphA47) (7.5 µM) for 10 min (37°C) (i.e. Stage I). Cells were then exposed to PMA (500 nM) (Stage II) for another 10 min, and endocytosis of Tf was measured. ( C ) L. major (5×10⁶) were incubated with varying concentrations of Tyrphostin A47 for 10 min. Parasites were rinsed, and endocytosis of Tf measured (see legend to Fig. 1 for protocol). ( D ) L major (5×10⁶/ml) was treated with Tyrphostin A47 (5 µM) for 15 min in culture medium. Thereafter, cells were incubated with PMA (500 nM; final conc.) for 15 min, and endocytosis of Tf was measured as described the legend to Figure 1.

Figure 8. A Working Model for DAG Activation of Tf Endocytosis in Trypanosomatids.

Based on our biochemical (Figs. 1 through Fig. 6), bioinformatic (Fig. 6, Table 1), and pharmacological data (Fig. 7), we propose that DAG binds to a C1_5 domain of a PTK in T. brucei (or L. major). The trypanosomatid PTK is activated by DAG, and the enzyme phosphorylates components of the endosomal pathway to activate uptake of Tf.