Proteomic analysis of CDK and cyclin complexes in Arabidopsis

Cyclin dependent kinases (CDKs) are threonine-serine kinases that, in complex with cyclins, regulate the progression of cell division in eukaryotic organisms. There are 14 CDKs and 49 cyclins in Arabidopsis, but the pairings between them are not well understood, and the substrates of these complexes remain largely unknown. To better understand the various CDK-cyclin interactions and to ascertain the substrates of these kinases, two different methods were used to isolate interacting proteins from Arabidopsis. Isolations of CDKA and CDKB complexes were carried out through immunoaffinity purifications using antisera specific to either type of CDK. A two-step tandem affinity purification (TAP) method was used to purify CYCD3;1 complexes. Subsequent tandem mass spectrometric analysis of purified proteins identified 51 putative interactors from duplicate purifications by CDKA and 63 from duplicate purifications by CDKB. Both CDKA and CDKBs interact with ribosomal proteins, though the overlap is very small, suggesting the two types of CDKs have distinct functions. Of the proteins pulled down with CDKA, 24% are involved in unknown biological processes, indicating that CDKA may be involved in cellular processes not yet elucidated. From CDKB purifications, 51% and 32% of the proteins identified are involved in protein metabolism and cell organisation, respectively, suggesting a role for CDKBs in protein degradation, regulation of the cytoskeleton, and organisation of microtubules. Surprisingly, CYCD4;1 was the only cyclin co-purified with CDKA. Moreover, two proteins were identified to be binding to CYCD3;1-TAP. CDKA was found to be the exclusive partner for CYCD3;1 and the two proteins are potentially in complex with heat shock protein 70. Both methods are successful in purifying CDK-cyclin complexes from Arabidopsis and therefore provide a tool to explore the composition of these complexes and to identify putative targets. Together with further validation, these purification strategies can provide insight into control of the cell cycle and other mechanisms by CDK-cyclin kinases.EThOS - Electronic Theses Online ServiceGBUnited Kingdo