Phosphorylation of Serine 148 in Giardia lamblia End‐binding 1 Protein is Important for Cell Division

Abstract

Giardia lamblia is a unicellular organism, showing a polarity with two nuclei and cytoskeletal structures. Accurate positioning of these organelles is essential for division of G. lamblia, which is poorly understood. Giardia lamblia end-binding 1 (GlEB1) protein and G. lamblia aurora kinase (GlAK) have been shown to modulate microtubule (MT) distribution during cytokinesis. A direct association between GlEB1 and GlAK was demonstrated. Like GlEB1, GlAK was also found at nuclear envelopes and median bodies of G. lamblia. In vitro kinase assays using Giardia lysates immunoprecipitated with anti-GlAK antibodies or recombinant GlAK suggested that GlEB1 is a substrate of GlAK. Site-directed mutagenesis indicated that threonine-205 in GlAK was auto-phosphorylated and that GlAK phosphorylated serine (Ser)-148 in GlEB1. Ectopic expression of a mutant GlEB1 (with conversion of Ser-148 into alanine of GlEB1) resulted in an increased number of Giardia cells with division defects. Treatment of G. lamblia with an AK inhibitor triggered cytokinesis defects, and ectopic expression of a phospho-mimetic mutant GlEB1 (with conversion of Ser-148 into aspartate) rescued the defects in Giardia cell division caused by the AK inhibitor. These results suggested that phosphorylation of GlEB1 played a role in cytokinesis in G. lamblia.

Roles of end‐binding 1 protein and gamma‐tubulin small complex in cytokinesis and flagella formation of Giardia lamblia

“…Therefore, we examined the phenotype of the GlEB1 knockdown cells with respect to the formation of the flagella and median body in the following experiments. A previous study indicated that knockdown of the GlEB1 resulted in a cytokinesis defect (Kim et al, ). GlEB1 knockdown also resulted in an increased proportion of Giardia cells without a median body (from 24% to 30% p ‐value = 0.0012; Figure a(i)).…”

“…The full length of recombinant GST‐tagged GlEB1 (GST‐GlEB1) protein was expressed in Escherichia coli BL21 (DE3) carrying pGEXEB1 1–238 (Kim et al, ) with 0.5 mM isopropyl β‐ᴅ‐1‐thiogalactopyranoside (IPTG) and purified using glutathione Sepharose ® 4B affinity chromatography (GE Healthcare, Uppsala, Sweden). GST protein was also purified as described above and used as a control.…”

“…Twenty micrograms of the eluted fraction was analyzed by western blotting using anti‐HA (Sigma‐Aldrich) to monitor the presence of HA‐tagged Glγ‐tubulin. The same sample was treated with antibodies against Glγ‐tubulin, GlEB1 (Kim et al, ), GlGCP2, or GlGCP3 antibodies. As a control, Giardia caring HA‐tagged Glγ‐tubulin extracts were incubated with anti‐mouse IgG conjugated to Sepharose ® beads (Cell Signaling, Danvers, MA) instead of anti‐HA agarose beads.…”

“…Next, the blot was incubated with horseradish peroxidase-conjugated secondary antibodies and the immunoreactive protein was detected using an enhanced chemiluminescence system (Thermo Scientific, Rockford, IL). The blots were then incubated in a stripping buffer (Thermo Scientific) for 30 min and reacted with polyclonal rat antibodies specific to the protein disulfide isomerase (PDI) 1 of G. lamblia (1:10,000) (Kim et al, 2017). cells were lysed by sonication with 2-s pulses at 20% amplitude (Pronextech, Seoul, Korea).…”

“…The same sample was treated with antibodies against Glγ-tubulin, GlEB1 (Kim et al, 2017), GlGCP2, or GlGCP3 antibodies. As a control, Giardia caring HA-tagged Glγ-tubulin extracts were incubated with anti-mouse IgG conjugated to Sepharose ® beads (Cell Signaling, Danvers, MA) instead of anti-HA agarose beads.…”