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.
Highlights
The protozoan parasite Leishmania is responsible for a wide spectrum of human diseases that cause varying degrees of patient morbidity and mortality and affect more than 12 million people world-wide
At least two leishmanial ecto-protein kinases (PK) released by promastigotes were identified as follows: first, an ecto-PK that is shed constitutively and phosphorylates phosvitin; and second, an enzyme released by incubation with PK substrates that phosphorylates protamine sulfate
Most cells examined appear to release cyclic nucleotide independent casein kinases (CK), the release of protein kinase (PKA) or protein kinase C (PKC) has been documented in only a few cases
Summary
Surface PK (ecto-PK), the potential for their involvement in signal transduction and cell-cell interactions appears great. We have identified a cyclic nucleotide-independent ecto-PK activity on viable Leishmania major promastigotes that phosphorylate exogenous substrates, such as mixed histones and protamine sulfate, in addition to 11 endogenous parasite membrane proteins [12]. The inducible release or shedding of ecto-PK in the presence of enzyme substrates has been described for specific PK on the surface of HeLa cells, endothelial cells, fibroblasts, neutrophils, and other cells [6, 7, 9, 15, 16] This activity appears to be similar to casein kinases and was recently purified from HeLa cells and characterized as casein kinase 1 (CK1) and casein kinase 2 (CK2, see Ref. 6). These findings will allow us to further characterize the properties and roles of ecto-PK and the possible ramifications of these enzymes on host-parasite interactions
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