G CRK3 activity t, As is the case for CDC28. L. personnel CRKs has have 12 and 10 thereof to maintain the T-loop Thr or Ser. To assess whether other CRKs of GST CIV1, L. could be phosphorylated TCR Pathway Important CRKs first Were cloned into pET15b and August and expressed from E. coli. CRK5 was not considered since it was reclassified as MOK MAP kinase family, and it is unlikely that-dependent cyclin-dependent. L. CRKs were hlt as important L. weight Mexicana genome was not available for analysis at the time and the CRK family in this type was unknown. Only L. Important CRK3his proved to be phosphorylated by GST CIV1. CRKs The purified monomers were tested for histone H1 kinase activity of t, but none were active.
These data show that yeast CIV1 GST CRK3 specificity t For Leishmania CRK with the gr Th homology to CIV1, s natural substrate, CDC28 and CRKs Leishmania not active histone H1 kinase in L Expressed soluble protein monomers. It is however not exclude En based its activation by cyclin partner as yet unidentified activity or t as monomers with other substrate. 3.2 An active CRK3: CYCA complex L. Major CYCA was amplified with a C-or N-terminal HA tag, and generate an expression vector for episomal pGL1388 PXG and pGL1389. Promastigotes of L. The employees were each plasmid and transfected cell lines resistant to G418 alone. The expression of both HA and HA CYCA CYCA was detected in cell lysates procyclic promastigotes the predicted size S of 35 kDa, whereas no significant HA protein was detected in wild-type cells. Immuno F Filling of L. major and L.
rod was carried out by using a S Molecules conjugated Antique Body against HA. Proteins Immunopr from cell lysates Zipitiert separated by SDS-PAGE and with silberf Staining angef Rbt. A protein corresponding to the expected size S of HA tagged CYCA was from L. immunpr Zipitiert Important, but not wild-type L. Staff. CRK3 was with a specific antibody Zipitaten body in Immunpr Detected CRK3 L. Important, but not wild-type L. employee best Term that CRK3 CYCA interacts with promastigotes in procyclical. The precipitated material was for histone H1 kinase activity T tested. The activity T been in immuno Pr Zipitate L. detected Important, but not wild-type L. Staff. These data show that with CYCA CRK3 active form in vivo and histone H1 kinase interacts.
4th Discussion The present work is the first to describe the production of a defined active recombinant CRK3 kinase complex, and show that, even if the stock CDK ugetieren Leishmania certain regulatory functions with CDK S Yeast and there are also important differences. In this study, l Soluble CRK3 expressed in bacteria, purified, and was histone H1 kinase possess negligible Ssigbar. Putative cyclin, CYCA, was identified L. mexicana and expressed in bacteria. The purified protein was found to bind and activate CYCA CRK3 in vitro dose-dependent-Dependent manner, with a kinase activity occurs T if the optimal molar Ratio of cyclin-kinase was at 1:1. The syntenic counterpart CYCA in L. donovani LdCYC1 has already shown LdCRK3 bind in vivo, but could be misfolded LdCRK3 to activate by the bacteria in vitro, probably due to the recombinant protein expressed, and therefore inactive.