Retraction Retraction: Mutational analysis of the phosphorylation sites of the Leishmania mexicana kinesin homologue LmxKin29 ( J.

s Kinesins are motor proteins, have been identified in a wide diversity of eukaryotes, from protists to mammals. The kinetoplastids‟ genome sequences have also shown the presence of a large number of kinesins. The current study presents characterise a novel L.mexicana kinesin LmxM29.3. 050, thought to be associated with flagellum formation. It was found express in both the amastigote and promastigote life stages. In this study we assigned this protein as a kinesin homologue to the unknown or orphan kinesins superfamily (1). The biochemical analysis showed the MAP kinase homologue LmxMPK3 can phosphorylate full length of LmxKin29 at serine 554. Localisation studies using GFP-tagged LmxKin29 revealed that it is predominantly found in between the nucleus and the flagellar pocket, while in dividing cells LmxKin29 was found at the anterior and posterior ends of the cells. Hence, LmxKin29 might play a role in cytokinesis. Double allele deletion was successfully generated. Morphological analysis of promastigotes with LmxKin29 tagged with GFP displayed no obvious phenotypic differences comparing the mutants with wild type cells. The eluted and dialysed fusion proteins 2-5 µg/µL of kinase assay solution(5 µL 10 × kinase buffer, 2.5 μL of cpm/pmol [γ -32P] ATP and ddH2O were added to a total of µL) . As a control substrate 5 μL MBP 4.5 μg/μL was used. All reaction tubes were placed in an end-over- end rotator and incubated at 27°C for one hour. 12.5 μL of 5 × SSB 2% (SDS, 20% glycerol, 0.001% bromphenol blue, 200 mM DTT and 62.5 mM Tris-HCl w ere added, the sample heated in a heat block at 95∞C for 10 minutes and subsequently resolved on SDS-PAGE. The gel was Coomassie-stained, dried and exposed to X-ray films at - 70∞C.The GST -tag by cleavage with thrombin if required. MAP kinase His-LmxMPK3 used in this study was expressed with an N-terminal hexa-histidine tag For in vitro analysis co-expression of hexahistidine-tagged LmxMPK3 along with its activating MAP kinase kinase LmxMKK was used (23). The expression vector pJCduet, contains two multiple cloning sites (MCSs). LmxMPK3 was cloned in the first one and the second MCS was used to clone the gene for the activator kinase LmxMKK (Appendix1). This construct allows purification activated His- LmxMPK3 from bacteria for both N- and C-terminally tagged kinesin. These results are consistent with the phenotype investigations of null mutants for LmxKin29 , which proved that LmxKin29 deficiency had no effect on the flagellum length, body width and body length of promastigotes. The proximal flagellar localisation has been shown to be an important regulatory site for different proteins in eukaryotic cells (32) like for instance a NIMA-related kinase, FA2, which is consistent with its role in deflagellation (33). We anticipated that LmxKin29 cellular similarity in localisation with another kinesin could give a clue about its function. Kinesin-13 (32), and Tb927.6. 1770 (29) have a similar localisation as LmxKin29 in dividing cells. Hence, this localisation provides evidence that LmxKin29 might be responsible for certain fundamental cellular functions such as microtubule organisation or may mediate cell division. This should be addressed in further work. superfamily. LmxKin29 from L . mexicana is a putative kinesin with a high sequence similarity to the L . major kinesin LmjF.30. 0350 that belongs to the “orphan” kinesins, introduced by (1). LmxKin29 also has a homologue in T. brucei (Tb927.5.1870) which was mentioned recently as an orphan kinesin . One of the unanticipated findings was that the LmxKin29 null mutant clones did not show any significant difference compared with the phenotype of the L. mexicana wild type. This result proved that LmxKin29 is not involved in the L. mexicana flagellum assembly. LmxKin29 plays a significant role in L. mexicana pathogenicity in vivo. The most important clinically relevant finding showed that LmxKin29 null mutants are unable to cause lesions in infected Balb/c mice and that no parasites could be detected at the injection site ten weeks post infection. LmxKin29 is not the only kinesin that is essential for parasite survival in an infected host. LmxKin29 and TbKif13-1 have a similar localisation in dividing cells and they both affect the parasite‟s pathogenicity. LmxKin29 is essential for survival of the amastigote parasite offering that it would be an ideal drug target.