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.

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 ⌺1278b 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 human family of MAP 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 MAP kinase-binding site, Mnk1b shows higher basal activity than Mnk1a. In contrast, the activity of Mnk1a is tightly regulated by signalling through ERK and p38 MAP kinase.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 isozyme while allowing both properties to be stimulated by upstream MAP kinase signalling.Two features of the C-terminus of Mnk1a appear to account for this behaviour: the known MAP kinase-binding site and a region (predicted to be -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 identify a conserved phenylalanine 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.Keywords: protein kinase; Mnk; ERK; p38 MAP kinase; eIF4E Abbreviations used: eIF, eukaryotic initiation factor; ERK, extracellular ligand-regulated kinase; HEK, human embryonic kidney; hMnk, human Mnk; MAP kinase, mitogen-activated protein kinase; mMnk, mouse Mnk; Mnk, MAP kinase signal-integrating kinase (or MAP kinase-interacting kinase); NES, nuclear export sequence; NLS, nuclear localisation sequence. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited IntroductionThe MAP kinase signal-integrating kinases (also termed 'MAP kinase-interacting' kinases; Mnks) were first discovered by virtue of their abilities to be phosphorylated by [1] or to bind to [2] ERK and/or p38 MAP kinase. There are two Mnk genes in mice and humans, Mnk1 and Mnk2. The Mnk proteins that were originally reported (here termed Mnk1a and Mnk2a) each contain, within their C-terminal regions, a motif for binding to MAP kinases (Fig. 1A). Mnk1a binds well to both ERK and p38 MAP kinases ( / ) while Mnk2a binds better to ERK and only weakly to p38 MAP kinases / [2;3]. ERK and p38 MAP kinases phosphorylate two threonine residues in Mnk1/2 which are located in the activation or 'T'-loop. In many protein kinases phosphorylation in this region leads to their activation.Subsequently it was shown that the human Mnk1 and Mnk2 genes each give rise to two mRNAs, and thus to two distinct polypeptides, as a consequence of alternative mRNA splicing [4][5][6][7] (Fig. 1A). In each case, the second form of each polypeptide (Mnk1b or Mnk2b) contains a distinct C-termina...

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