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Release of ecto-protein kinases by the protozoan parasite Leishmania major
N Sacerdoti-Sierra1, C L Jaffe
1Department of Parasitology, The Kuvin Centre for the Study of Infectious and Tropical Diseases, Hebrew University-Hadassah Medical School, P. O. Box 12272, Jerusalem 91120, Israel.
Abstract:
Leishmania major promastigotes have externally oriented ecto-protein kinases (PK) that are capable of phosphorylating both endogenous membrane substrates and foreign proteins. Live parasites phosphorylate protamine sulfate, casein, and phosvitin but not bovine serum albumin. Addition of exogenous PK substrates, such as phosvitin or casein, induced the shedding of ecto-PK that are capable of phosphorylating protamine sulfate. No phosphorylation of protamine sulfate was seen when cell-free supernatants from promastigotes incubated with either buffer alone or bovine serum albumin were used. A second enzyme, a constitutively released PK that phosphorylates casein or phosvitin and not protamine sulfate or mixed histones, was identified and characterized. This PK is inhibited by 5 microM staurosporine, 50 microg/ml heparin, and 75 microM CKI-7, concentrations typical of the IC50 found for other eukaryotic casein kinases (CK). The constitutively shed ecto-PK specifically phosphorylated a peptide substrate for CK1 but not for CK2, suggesting that this shed PK is similar to CK1.
Insights
Leishmania major parasites release external protein kinases (PK). These enzymes phosphorylate substrates, and their release can be triggered by specific substrates, suggesting a regulatory mechanism.
Area of Science:
- Parasitology
- Molecular Biology
- Biochemistry
Background:
- Leishmania major promastigotes possess externally oriented ecto-protein kinases (PK).
- These ecto-PKs can phosphorylate both endogenous membrane substrates and foreign proteins.
- The functional role and regulation of these extracellular enzymes are not fully understood.
Purpose of the Study:
- To investigate the phosphorylation capabilities of Leishmania major ecto-protein kinases.
- To determine if exogenous substrates influence the shedding of ecto-PKs.
- To characterize a constitutively released PK and compare it to known kinases.
Main Methods:
- Incubation of live Leishmania major promastigotes with various protein substrates (protamine sulfate, casein, phosvitin, bovine serum albumin).
- Analysis of phosphorylation activity in cell-free supernatants after incubation with different conditions.
- Characterization of a constitutively released PK using specific inhibitors (staurosporine, heparin, CKI-7) and peptide substrates for casein kinase 1 (CK1) and casein kinase 2 (CK2).
Main Results:
- Live parasites phosphorylated protamine sulfate, casein, and phosvitin, but not bovine serum albumin.
- Exogenous substrates like phosvitin or casein induced the shedding of ecto-PKs capable of phosphorylating protamine sulfate.
- A second, constitutively released PK was identified, which phosphorylates casein/phosvitin but not protamine sulfate/histones, and is inhibited by staurosporine, heparin, and CKI-7, suggesting it is similar to CK1.
Conclusions:
- Leishmania major ecto-protein kinases exhibit substrate-dependent phosphorylation and shedding.
- The shedding of specific ecto-PKs can be modulated by the presence of exogenous substrates.
- A constitutively released ecto-PK, likely a form of CK1, plays a role in extracellular enzymatic activity.