Cdk8 regulates stability of the transcription factor Phd1 to control pseudohyphal differentiation of Saccharomyces cerevisiae.

The yeast Saccharomyces differentiates into filamentous pseudohyphae when exposed to a poor source of nitrogen in a process involving a collection of transcription factors regulated by nutrient signaling pathways. Phd1 is important for this process in that it regulates expression of most other transcription factors involved in differentiation and can induce filamentation on its own when overproduced. In this article, we show that Phd1 is an unstable protein whose degradation is initiated through phosphorylation by Cdk8 of the RNA polymerase II mediator subcomplex. Phd1 is stabilized by cdk8 disruption, and the naturally filamenting Σ1278b strain was found to have a sequence polymorphism that eliminates a Cdk8 phosphorylation site, which both stabilizes the protein and contributes to enhanced differentiation. In nitrogen-starved cells, PHD1 expression is upregulated and the Phd1 protein becomes stabilized, which causes its accumulation during differentiation. PHD1 expression is partially dependent upon Ste12, which was also previously shown to be destabilized by Cdk8-dependent phosphorylations, but to a significantly smaller extent than Phd1. These observations demonstrate the central role that Cdk8 plays in initiation of differentiation. Cdk8 activity is inhibited in cells shifted to limiting nutrient conditions, and we argue that this effect drives the initiation of differentiation through stabilization of multiple transcription factors, including Phd1, that cause activation of genes necessary for filamentous response.

The C-terminal domain of Mnk1a plays a dual role in tightly regulating its activity.

The human family of MAPK (mitogen-activated protein kinase) signal-integrating kinases (Mnks) comprises four related proteins derived from two genes by alternative splicing. The MNK1 gene gives rise to two proteins, Mnk1a and Mnk1b, which possess distinct C-termini and properties. Despite lacking the C-terminal MAPK-binding site, Mnk1b shows higher basal activity than Mnk1a. In contrast, the activity of Mnk1a is tightly regulated by signalling through ERK (extracellular-signal-regulated kinase) and p38 MAPK. We show that the short C-terminus of Mnk1b confers on it a 'default' behaviour of substantial, but unregulated, activity. In contrast, the longer C-terminus of Mnk1a represses the basal activity and T (activation)-loop phosphorylation of this isoenzyme while allowing both properties to be stimulated by upstream MAPK signalling. Two features of the C-terminus of Mnk1a appear to account for this behaviour: the known MAPK-binding site and a region (predicted to be alpha-helical) which occludes access to the catalytic domain and the T-loop. The activation of Mnk1a results in a marked conformational change leading to a more 'open' structure. We also identified a conserved phenylalanine residue in an Mnk-specific insert as playing a key role in governing the ease with which Mnk1a can be phosphorylated. These studies help to identify the features that give rise to the diverse properties of human Mnk isoforms.

Srb10/Cdk8 regulates yeast filamentous growth by phosphorylating the transcription factor Ste12.

The budding yeast Saccharomyces cerevisiae differentiates into filamentous invasively growing forms under conditions of nutrient limitation. This response is dependent on the transcription factor Ste12 and on the mating pheromone-response mitogen-activated protein (MAP) kinase cascade, but a mechanism for regulation of Ste12 by nutrient limitation has not been defined. Here we show that Ste12 function in filamentous growth is regulated by the cyclin-dependent kinase Srb10 (also known as Cdk8), which is associated with the RNA polymerase II holoenzyme. Srb10 inhibits filamentous growth in cells growing in rich medium by phosphorylating Ste12 and decreasing its stability. Under conditions of limiting nitrogen, loss of Srb10 protein and kinase activity occurs, with a corresponding loss of Ste12 phosphorylation. Mutation of the Srb10-dependent phosphorylation sites increases pseudohyphal development but has no effect on the pheromone response of haploid yeast. Srb10 kinase activity is also regulated independently of the mating pheromone-response pathway. This indicates that Srb10 controls Ste12 activity for filamentous growth in response to nitrogen limitation and is consistent with the hypothesis that Srb10 regulates gene-specific activators in response to physiological signals to coordinate gene expression with growth potential.