title: Autophosphorylation at Thr279 of Entamoeba histolytica atypical kinase EhAK1 is required for activity and regulation of erythrophagocytosis.
abstract: Phagocytosis plays a key role in survival and pathogenicity of Entamoeba histolytica. We have recently demonstrated that an atypical kinase EhAK1 is involved in phagocytosis in this parasite. It is recruited to the phagocytic cups through interaction with EhCaBP1. EhAK1 manipulates actin dynamics by multiple mechanisms including phosphorylation of G-actin. Biochemical analysis showed that EhAK1 is a serine/threonine kinase with broad ion specificity and undergoes multiple trans-autophosphorylation. Three autophosphorylation sites were identified by mass spectrometry. Out of these Thr279 appears to be involved in both autophosphorylation as well as substrate phosphorylation. Over expression of the mutant Thr279A inhibited erythrophagocytosis showing dominant negative phenotype. Multiple alignments of different kinases including alpha kinases displayed conserved binding sites that are thought to be important for function of the protein. Mutation studies demonstrated the importance of some of these binding sites in kinase activity. Binding studies with fluorescent-ATP analogs supported our prediction regarding ATP binding site based on sequence alignment. In conclusion, EhAK1 has multiple regulatory features and enrichment of EhAK1 at the site of phagocytosis stimulates trans-autophosphorylation reaction that increases kinase activity resulting in enhanced actin dynamics and phagocytosis. Some of the properties of EhAK1 are similar to that seen in alpha kinases.
authors.affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India.
authors.forename: M Shahid
authors.initials: MS
authors.lastname: Mansuri
authors.affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India.
authors.forename: Mrigya
authors.initials: M
authors.lastname: Babuta
authors.affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India.
authors.forename: Mohammad Sabir
authors.initials: MS
authors.lastname: Ali
authors.affiliation: School of Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India.
authors.forename: Ravi
authors.initials: R
authors.lastname: Bharadwaj
authors.affiliation: National Institute of Immunology, New Delhi 110067, India.
authors.forename: Gagan
authors.initials: G
authors.lastname: Deep jhingan
authors.affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India.
authors.forename: Samudrala
authors.initials: S
authors.lastname: Gourinath
authors.affiliation: School of Environmental Sciences, Jawaharlal Nehru University, New Delhi 110067, India.
authors.forename: Sudha
authors.initials: S
authors.lastname: Bhattacharya
authors.affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India.
authors.forename: Alok
authors.initials: A
authors.lastname: Bhattacharya
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D057899:Cytophagocytosis; D004748:Entamoeba histolytica / Q000502:physiology*; D004789:Enzyme Activation; D004912:Erythrocytes; D010766:Phosphorylation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000255:Adenosine Triphosphate; D011494:Protein Kinases