Analysis of the role of the C-terminus of LmxMPK6

LmxMPK6 is one of only two L. mexicana MAP kinase homologues with an extremely prolonged carboxy-terminus (Wiese, M. et al. 2003b). Within the C-terminus of LmxMPK6 four possible SH3-binding sites and two possible nuclear localisation signals have been identified (Wiese, M. et al. 2003b). SH3-motifs constitute binding sites for proline-rich peptides of, for instance, scaffold proteins or regulatory proteins (Engstrom, W. et al.

2010; Scott, J. D. et al. 2009). C-terminal extensions in other kinases have been found to play various roles. The C-terminus of ERK7 is required for kinase activity and for the subcellular localization of the MAP kinase (Abe, M. K. et al. 1999). The kinase activity of ERK5 on the other hand was greatly increased by the truncation of its C-terminal extension, but its nucleocytoplasmic shuttling was equally impaired (Buschbeck, M. et al.

2005). The prolonged C-terminus of TbECK1, the T. brucei homologue of LmxMPK6, has been suggested to have a negative autoregulatory function on the kinase as its absence leads to an aberrant activity of the truncated TbECK1 and consequently to abnormal phenotypes (Ellis, J. et al. 2004). To investigate the function of the C-terminal extension of LmxMPK6, two different truncated versions of the protein were created. The mutant LmxMPK6short was truncated directly after the kinase domain of LmxMPK6, while LmxMPK6short2TY additionally contained 78 amino acids of the C-terminus and thereby corresponded to the truncated mutant of TbECK1 used by Ellis et al. (Ellis, J. et al. 2004).

The recombinant expression of both truncated mutants as GST-fusion proteins in E. coli yielded reasonable quantities of rather pure protein and was therefore drastically superior to the recombinant expression of the full-length GST-LmxMPK6. This confirms the notion that the poor recombinant expression of GST-LmxMPK6 and GST-LmxMPK6K33M is due to the large size of the proteins, as discussed earlier. GST-LmxMPK6short did not display any phosphotransferase activity towards MBP but a slight amount of autophosphorylation (Fig. 52, lanes 3, 3’). GST-LmxMPK6short2TY on the other hand phosphorylated MBP significantly and also displayed a prominent autophosphorylation (Fig. 52, lanes 4, 4’).

This finding demonstrates that the additional 78 amino acids of the C-terminal extension which are present in LmxMPK6short2TY are essential for kinase activity. Despite the fact that the α-helix usually present in kinases C-terminal of the kinase domain had not been predicted for LmxMPK6, this region still played an indispensable role for the kinase activity of LmxMPK6 and likely does contain the α-helix. The phosphotransferase activity of GST-LmxMPK6short2TY towards MBP showed no consistent results in comparison with kinase activity levels of full-length GST-LmxMPK6. This is most likely at least partially due to the difficulty in judging protein levels of GST-LmxMPK6 contained in the kinase assays, owing to its extremely low abundance in protein eluates. No statement can therefore be made regarding the phosphotransferase activity of GST-LmxMPK6short2TY in comparison to the wild type protein GST-LmxMPK6 which contained the full C-terminal extension. It is

however striking that full-length GST-LmxMPK6 does not display any auto-phosphorylation, while both truncated mutants autophosphorylate. This can be viewed as an indicator for the enhanced activity of GST-LmxMPK6short2TY. The findings are in accordance with properties of ERK5. Truncated versions of ERK5 showed higher auto-phosphorylation than the wild type protein ERK5, but autoauto-phosphorylation decreased with increasing truncation of the protein (Buschbeck, M. et al. 2005). ERK5 was intriguingly also able to autophosphorylate within its C-terminus (Mody, N. et al. 2003).

To summarise, the truncation of LmxMPK6 at 78 amino acids after the end of its kinase domain led to the generation of LmxMPK6short2TY which could be recombinantly expressed to high quantities of pure protein and displayed a distinct phosphotransferase activity towards MBP as well as autophosphorylation activity. The 78 amino acids remaining of the C-terminus were required for the observed activity.

As LmxMPK6short2TY was constitutively active in vitro it was employed for in vivo investigations. The full-length protein TbECK1, the T. brucei homologue of LmxMPK6, was expressed from a tetracycline-inducible plasmid in T. brucei procyclics, which did not result in a distinct phenotype. When the truncated mutant of TbECK1 was extra-chromosomally expressed against an endogenous background expression of the full-length protein procyclic T. brucei cells displayed reduced growth and a high abundance of aberrant karyotypes. As the aberrant phenotype only emerged when the truncated mutant of TbECK1 was active, it was proposed that the C-terminus of TbECK1 has a negative autoregulatory function on the kinase (Ellis, J. et al. 2004). The extrachromosomal expression of LmxMPK6short2TY, the active truncated mutant of LmxMPK6, in the presence of genomic expression of endogenous full-length LmxMPK6 did not lead to any visible phenotype (Fig. 54). Neither did cells display delayed growth nor did they have a vast number of aberrant karyotypes. This indicates that the C-terminus in LmxMPK6 possibly does not play the same autoregulatory role as in its T. brucei homologue.

However, it is also very likely that the observation of no phenotype results from the experimental approach used. Unlike for the TbECK1 analysis in T. brucei, the extra-chromosomal expression of LmxMPK6short2TY was not inducible by tetracycline. The transformation of promastigotes with pXpolPac MPK6short2TY and subsequent selection of positive clones could therefore select cells which are able to compensate the aberrant phenotypes induced by the presence of irregularly active LmxMPK6short2TY.

Compensation could take place by suppression of plasmid numbers, by the inhibition of extrachromosomal expression or by degradation of the aberrantly active protein. The fact that attempts to detect extrachromosomal expression of LmxMPK6short2TY by immunoblot analysis using antiserum against the N-terminal end of LmxMPK6 (data not shown) failed, could be consistent with a lack of LmxMPK6short2TY in cells due to

compensation, but is by no means proof. Unfortunately, it was not possible within the scope of this thesis to investigate further, but future research would have to generate L. mexicana cell lines which allow tetracycline-inducible expression of proteins, as has been done for L. chagasi and L. donovani respectively (Yan, S. et al. 2002; Yao, C. et al.

2007).

As it was not possible to deduce any negative autoregulatory function of the C-terminal extension in vivo and the comparison of the in vitro kinase activity of full-length GST-LmxMPK6 with truncated GST-GST-LmxMPK6short2TY was challenging, the effect of the C-terminus was investigated with the help of the pJCduet co-expression system. The poor expression and purification of full-length GST-LmxMPK6 were most likely arising from the large size of the protein. Therefore, the C-terminus and N-terminus of LmxMPK6 were cloned separately. With the help of the pJCduet co-expression system (John von Freyend, S. et al. 2010) it was possible to analyse the influence of the C-terminus on the kinase activity of the N-terminus. The recombinant expression and tag purification of His-LmxMPK6Nterm yielded high levels of pure, active recombinant protein (Fig. 57), which was considerably more active than GST-LmxMPK6short2TY (Fig. 57 and Fig. 58). The only difference between recombinant GST-LmxMPK6short2TY and His-LmxMPK6Nterm was in the choice of tags with which the proteins were expressed. This indicates that the GST-tag and possibly also the much smaller TY-tag have a negative impact on the expression levels and purification results of LmxMPKshort2, as well as on the phosphotransferase activity of LmxMPK6short2 to a certain extent. This finding is contrary to the enhanced expression of GST-LmxMPK4 in comparison to His-LmxMPK4 (4.1.2.2), which demonstrates that the choice of tag for recombinant expression must be validated independently for every protein. It can therefore be expected that the expression of full-length LmxMPK6 should also yield higher levels of protein after recombinant expression as a His-tag protein. It is nevertheless likely that the poor expression of LmxMPK6 is also due to the large size of the protein which can lead to premature abortion of translation and subsequent degradation of the protein. The co-expression of His-LmxMPK6Nterm with S-LmxMPK6Cterm had no definite effect on the kinase activity of His-LmxMPK6Nterm. A slight reduction in kinase activity was visible after the co-expression with S-LmxMPK6Cterm, but this could quite likely result from variations in the assay (Fig. 57).

It is therefore unlikely that the C-terminus of LmxMPK6 has a negative autoregulatory function towards the kinase activity of LmxMPK6 in vitro, contrary to ERK5 in which the presence of the C-terminus has a negative effect on in vitro phosphotransferase activity (Buschbeck, M. et al. 2005). However, it can not be excluded that in Leishmania the C-terminus plays a role in regulation of LmxMPK6, as has been shown for the tyrosine kinases c-Src (Bjorge, J. D. et al. 2000), c-Abl (Pluk, H. et al. 2002), Bcr-Abl (Smith, K. M.

et al. 2003) and the T. brucei homologue TbECK1 (Ellis, J. et al. 2004). The influence of

the C-terminus of TbECK1 on its in vitro kinase activity has not been investigated by Ellis et al., which is why it is still conceivable that TbECK1 and LmxMPK6 share the same regulatory mechanism. A regulatory influence of the C-terminus could be due to the binding of other proteins to the SH3-domains of the C-terminus changing its structure or to the translocation of the kinase into the nucleus. The translocation into the nucleus is somewhat unlikely as transcriptional control plays only a marginal role in kinetoplastids.