IDENTIFYING SUBSTRATES OF CYCLIN DEPENDENT KINASES BY SUBSTRATE TRAP & PURIFICAT

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We are seeking to understand how cyclins and cyclin-dependent kinases control the events of mitosis. In previous work, we found that a protein kinase called Gin4 is activated as cells enter mitosis and is required for the proper induction of specific mitotic events by the cyclin Clb2. To learn more about Gin4 function during mitosis, we have used a combination of genetics and biochemistry to identify proteins that functionally interact with Gin4. Both of these approaches have identified members of the septin family of proteins as playing a key role in the mitosis-specific activation of Gin4. Loss of septin function produces a phenotype that is very similar to loss of Gin4 function, and the mitosis-specific activation of Gin4 is dependent upon septin function. The septins are likely to play a direct role in the mitosis-specific activation of Gin4 because they bind tightly to Gin4 and are co-localized with Gin4 within the cell. These results demonstrate that the septins carry out functions early in mitosis before cytokinesis occurs, and that the septins may play a role in controlling mitotic signaling events. The elongated bud phenotype observed for septin mutations suggests that the septins function in the pathway used by Clb2, Nap1, and Gin4 to control bud growth during mitosis. Further evidence for such a role has come from affinity chromatography experiments that we have carried out to identify proteins that functionally interact with the Gin4 kinase. For these experiments, we purify a full length Gin4-GST fusion protein from bacteria and couple it to beads to create a Gin4 affinity column. The Gin4 affinity column is loaded with a crude extract made from rapidly growing yeast cells, and after washing with buffer the column is eluted with a gradient of salt. A number of proteins bind to the Gin4 affinity column, but not to a GST control column. To begin to characterize the proteins that bind to Gin4, we have used mass spectrometry to identify the major proteins that elute from the affinity column. We found that one of these proteins is Nap1, as expected from previous experiments in which we demonstrated a tight and specific interaction between Gin4 and Nap1. The other major proteins are members of the septin family, including Cdc3, Cdc10, Cdc11, Cdc12, and a new member of the septin family identified by the yeast genome sequencing project that we have named Sep5 (ORF designation YDL225W). Western blotting confirmed that Cdc11 binds to the column and that it is almost quantitativley depleted from the extract as it passes over the Gin4-GST column. Note that the septins co-elute, suggesting that they associate with each other as a complex in the same way that has been observed for Drosophila septins. When a crude extract from a Dnap1 strain is loaded onto a Gin4 affinity column the septins are still found to bind, demonstrating that Nap1 is not required for septin binding.