Serum-dependent selective expression of EhTMKB1-9, a member of Entamoeba histolytica B1 family of transmembrane kinases.
Entamoeba histolytica transmembrane kinases (EhTMKs) can be grouped into six distinct families on the basis of motifs and sequences. Analysis of the E. histolytica genome revealed the presence of 35 EhTMKB1 members on the basis of sequence identity (>or=95%). Only six homologs were full length containing an extracellular domain, a transmembrane segment and an intracellular kinase domain. Reverse transcription followed by polymerase chain reaction (RT-PCR) of the kinase domain was used to generate a library of expressed sequences. Sequencing of randomly picked clones from this library revealed that about 95% of the clones were identical with a single member, EhTMKB1-9, in proliferating cells. On serum starvation, the relative number of EhTMKB1-9 derived sequences decreased with concomitant increase in the sequences derived from another member, EhTMKB1-18. The change in their relative expression was quantified by real time PCR. Northern analysis and RNase protection assay were used to study the temporal nature of EhTMKB1-9 expression after serum replenishment of starved cells. The results showed that the expression of EhTMKB1-9 was sinusoidal. Specific transcriptional induction of EhTMKB1-9 upon serum replenishment was further confirmed by reporter gene (luciferase) expression and the upstream sequence responsible for serum responsiveness was identified. EhTMKB1-9 is one of the first examples of an inducible gene in Entamoeba. The protein encoded by this member was functionally characterized. The recombinant kinase domain of EhTMKB1-9 displayed protein kinase activity. It is likely to have dual specificity as judged from its sensitivity to different kinase inhibitors. Immuno-localization showed EhTMKB1-9 to be a surface protein which decreased on serum starvation and got relocalized on serum replenishment. Cell lines expressing either EhTMKB1-9 without kinase domain, or EhTMKB1-9 antisense RNA, showed decreased cellular proliferation and target cell killing. Our results suggest that E. histolytica TMKs of B1 family are functional kinases likely to be involved in serum response and cellular proliferation.
School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.
Shiteshu
S
Shrimal
Sudha
S
Bhattacharya
Alok
A
Bhattacharya
D000818:Animals; D017209:Apoptosis; D015152:Blotting, Northern; D015153:Blotting, Western; D016466:CHO Cells; D002448:Cell Adhesion; D049109:Cell Proliferation; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D016895:Culture Media, Serum-Free / Q000494:pharmacology*; D004748:Entamoeba histolytica / Q000201:enzymology* / Q000235:genetics*; D005455:Fluorescent Antibody Technique; D015723:Gene Library; D008156:Luciferases / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D010802:Phylogeny; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D016372:RNA, Antisense / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
D016895:Culture Media, Serum-Free; D008565:Membrane Proteins; D016372:RNA, Antisense; D012333:RNA, Messenger; D008156:Luciferases; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases