Matthias Peter

The F-box protein Skp2 is a ubiquitylation target of a Cul1-based core ubiquitin ligase complex: evidence for a role of Cul1 in the suppression of Skp2 expression in quiescent fibroblasts

The ubiquitin protein ligase SCFSkp2 is composed of Skp1, Cul1, Roc1/Rbx1 and the F‐box protein Skp2, the substrate‐recognition subunit. Levels of Skp2 decrease as cells exit the cell cycle and increase as cells re‐enter the cycle. Ectopic expression of Skp2 in quiescent fibroblasts causes mitogen‐independent S‐phase entry. Hence, mechanisms must exist for limiting Skp2 protein expression during the G0/G1 phases. Here we show that Skp2 is degraded by the proteasome in G0/G1 and is stabilized when cells re‐enter the cell cycle. Rapid degradation of Skp2 in quiescent cells depends on Skp2 sequences that contribute to Cul1 binding and interference with endogenous Cul1 function in serum‐deprived cells induces Skp2 expression. Furthermore, recombinant Cul1–Roc1/Rbx1–Skp1 complexes can catalyse Skp2 ubiquitylation in vitro. These results suggest that degradation of Skp2 in G0/G1 is mediated, at least in part, by an autocatalytic mechanism involving a Skp2‐bound Cul1‐based core ubiquitin ligase and imply a role for this mechanism in the suppression of SCFSkp2 ubiquitin protein ligase function during the G0/G1 phases of the cell cycle.

Nuclear sequestration of the exchange factor Cdc24 by Far1 regulates cell polarity during yeast mating.

Cytoskeletal rearrangements during the cell cycle and in response to signals are regulated by small Rho-type GTPases, but it is not known how these GTPases are activated in a spatial and temporal manner. Here we show that Cdc24, the guanine-nucleotide exchange factor for the yeast GTPase Cdc42, is sequestered in the cell nucleus by Far1. Export of Cdc24 to a site of cell polarization is mediated by two mechanisms. At bud emergence, activation of the G1 cyclin-dependent kinase Cdc28–Cln triggers degradation of Far1 and, as a result, relocation of Cdc24 to the cytoplasm. Cells overexpressing a non-degradable Far1 were unable to polarize their actin cytoskeleton because they failed to relocate Cdc24 to the incipient bud site. In contrast, in response to mating pheromones, the Far1–Cdc24 complex is exported from the nucleus by Msn5. This mechanism ensures that Cdc24 is targeted to the site of receptor-associated heterotrimeric G-protein activation at the plasma membrane, thereby allowing polarization of the actin cytoskeleton along the morphogenetic gradient of pheromone. Either degradation of Far1 or its nuclear export by Msn5 was sufficient for cell growth, suggesting that the two mechanisms are redundant for cell viability. Taken together, our results indicate that Far1 functions as a nuclear anchor for Cdc24. This sequestration regulates cell polarity in response to pheromones by restricting activation of Cdc42 to the site of pheromone receptor activation.

Phosphorylation of the MEKK Ste11p by the PAK-like kinase Ste20p is required for MAP kinase signaling in vivo.

BACKGROUNDnMany signals are transduced from the cell surface to the nucleus through mitogen-activated protein (MAP) kinase cascades. Activation of MAP kinase requires phosphorylation by MEK, which in turn is controlled by Raf, Mos or a group of structurally related kinases termed MEKKs. It is not understood how MEKKs are regulated by extracellular signals. In yeast, the MEKK Ste11p functions in multiple MAP kinase cascades activated in response to pheromones, high osmolarity and nutrient starvation. Genetic evidence suggests that the p21-activated protein kinase (PAK) Ste20p functions upstream of Ste11p, and Ste20p has been shown to phosphorylate Ste11p in vitro.nnnRESULTSnSte20p phosphorylated Ste11p on Ser302 and/or Ser306 and Thr307 in yeast, residues that are conserved in MEKKs of other organisms. Mutating these sites to non-phosphorylatable residues abolished Ste11p function, whereas changing them to aspartic acid to mimic the phosphorylated form constitutively activated Ste11p in vivo in a Ste20p-independent manner. The amino-terminal regulatory domain of Ste11p interacted with its catalytic domain, and overexpression of a small amino-terminal fragment of Ste11p was able to inhibit signaling in response to pheromones. Mutational analysis suggested that this interaction was regulated by phosphorylation and dependent on Thr596, which is located in the substrate cleft of the catalytic domain.nnnCONCLUSIONSnOur results suggest that, in response to multiple extracellular signals, phosphorylation of Ste11p by Ste20p removes an amino-terminal inhibitory domain, leading to activation of the Ste11 protein kinase. This mechanism may serve as a paradigm for the activation of mammalian MEKKs.

MAP kinase dynamics in response to pheromones in budding yeast

Although scaffolding is a major regulator of mitogen-activated protein kinase (MAPK) pathways, scaffolding proteins are poorly understood. During yeast mating, MAPK Fus3p is phosphorylated by MAPKK Ste7p, which is activated by MAPKKK Ste11p. This MAPK module interacts with the scaffold molecule Ste5p. Here we show that Ste11p and Ste7p were predominantly cytoplasmic proteins, while Ste5p and Fus3p were found in the nucleus and the cytoplasm. Ste5p, Ste7p and Fus3p also localized to tips of mating projections in pheromone-treated cells. Using fluorescence recovery after photobleaching (FRAP), we demonstrate that Fus3p rapidly shuttles between the nucleus and the cytoplasm independently of pheromones, Fus3p phosphorylation and Ste5p. Membrane-bound Ste5p can specifically recruit Fus3p and Ste7p to the cell cortex. Ste5p remains stably bound at the plasma membrane, unlike activated Fus3p, which dissociates from Ste5p and translocates to the nucleus.

Nuclear‐specific degradation of Far1 is controlled by the localization of the F‐box protein Cdc4

Far1 is a bifunctional protein that is required to arrest the cell cycle and establish cell polarity during yeast mating. Here we show that SCFCdc4 ubiquitylates Far1 in the nucleus, which in turn targets the multi‐ubiquitylated protein to 26S proteasomes most likely located at the nuclear envelope. In response to mating pheromones, a fraction of Far1 was stabilized after its export into the cytoplasm by Ste21/Msn5. Preventing nuclear export destabilized Far1, while conversely cytoplasmic Far1 was stabilized, although the protein was efficiently phosphorylated in a Cdc28–Cln‐dependent manner. The core SCF subunits Cdc53, Hrt1 and Skp1 were distributed in the nucleus and the cytoplasm, whereas the F‐box protein Cdc4 was exclusively nuclear. A cytoplasmic form of Cdc4 was unable to complement the growth defect of cdc4‐1 cells, but it was sufficient to degrade Far1 in the cytoplasm. Our results illustrate the importance of subcellular localization of F‐box proteins, and provide an example of how an extracellular signal regulates protein stability at the level of substrate localization.

The Cdc42p effector Gic2p is targeted for ubiquitin-dependent degradation by the SCFGrr1 complex

Cdc42p, a Rho‐related GTP‐binding protein, regulates cytoskeletal polarization and rearrangements in eukaryotic cells. In yeast, Gic1p and Gic2p are effectors of Cdc42p involved in actin polarization at bud emergence. Gic2p is expressed in a cell cycle‐dependent manner and rapidly disappears shortly after bud emergence concomitant with the activation of the G1 cyclin‐dependent kinase Cdc28p–Clnp. Here we have shown that the rapid disappearance of Gic2p results from ubiquitin‐dependent proteolysis. Biochemical and genetic evidence demonstrates that degradation of Gic2p required the Skp1–cullin–F‐box protein complex (SCF) components Cdc34p, Cdc53p, Skp1p and Grr1p, but not Cdc4p. Phosphorylation of several C‐terminal sites of Gic2p served as part of the recognition signal for ubiquitination. In addition, binding of Gic2p to Cdc42p was a prerequisite for degradation, suggesting that specifically the active form of Gic2p is targeted for destruction. Finally, our data indicate that degradation of Gic2p may be part of a mechanism which restricts cytoskeletal polarization in the G1 phase of the cell cycle.

Actin cytoskeleton organization regulated by the PAK family of protein kinases

Cdc42, Rac1 and other Rho-type GTPases regulate gene expression, cell proliferation and cytoskeletal architecture [1,2]. A challenge is to identify the effectors of Cdc42 and Rac1 that mediate these biological responses. Protein kinases of the p21-activated kinase (PAK) family bind activated Rac1 and Cdc42, and switch on mitogen-activated protein (MAP) kinase pathways; however, their roles in regulating actin cytoskeleton organization have not been clearly established [3-5]. Here, we show that mutants of the budding yeast Saccharomyces cerevisiae lacking the PAK homologs Ste20 and Cla4 exhibit actin cytoskeletal defects, in vivo and in vitro, that resemble those of cdc42-1 mutants. Moreover, STE20 overexpression suppresses cdc42-1 growth defects and cytoskeletal defects in vivo, and Ste20 kinase corrects the actin-assembly defects of permeabilized cdc42-1 cells in vitro. Thus, PAKs are effectors of Cdc42 in pathways that regulate the organization of the cortical actin cytoskeleton.

MAP Kinase Cascades: Scaffolding Signal Specificity

Scaffold proteins organize many MAP kinase pathways by interacting with several components of these cascades. Recent studies suggest that scaffold proteins provide local activation platforms that contribute to signal specificity by insulating different MAP kinase pathways.

The regulation of cyclin-dependent kinase inhibitors (CKIs)

Inhibitors of cyclin-dependent kinases (CKIs) play key roles in coordinating cell proliferation and development. They also function to control critical cell cycle transitions and as effectors of checkpoint pathways. The activity of CKIs is tightly controlled through the cell cycle and in response to various signals. Regulation generally affects the levels or availability of the CKIs rather than changing their intrinsic activities. Mechanisms controlling CKI function include the regulation of transcription, translation and proteolysis. In addition some signals appear to induce sequestration of CKIs within the cells, thereby changing their ability to interact with specific targets.

MAP kinase dynamics in yeast

Summry— MAP kinase pathways play key roles in cellular responses towards extracellular signals. In several cases, the three core kinases interact with a scaffold molecule, but the function of these scaffolds is poorly understood. They have been proposed to contribute to signal specificity, signal amplification, or subcellular localization of MAP kinases. Several MAP kinases translocate to the nucleus in response to their activation, suggesting that nuclear transport may provide a regulatory mechanism. Here we describe new applications for Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP), to study dynamic translocations of MAPKs between different subcellular compartments. We have used these methods to measure the nuclear/cytoplasmic dynamics of several yeast MAP kinases, and in particular to address the role of scaffold proteins for MAP‐kinase signaling.