Comprehensive characterization of the Hsp70 interactome reveals novel client proteins and interactions mediated by posttranslational modifications.

Hsp70 interactions are critical for cellular viability and the response to stress. Previous attempts to characterize Hsp70 interactions have been limited by their transient nature and the inability of current technologies to distinguish direct versus bridged interactions. We report the novel use of cross-linking mass spectrometry (XL-MS) to comprehensively characterize the Saccharomyces cerevisiae (budding yeast) Hsp70 protein interactome. Using this approach, we have gained fundamental new insights into Hsp70 function, including definitive evidence of Hsp70 self-association as well as multipoint interaction with its client proteins. In addition to identifying a novel set of direct Hsp70 interactors that can be used to probe chaperone function in cells, we have also identified a suite of posttranslational modification (PTM)-associated Hsp70 interactions. The majority of these PTMs have not been previously reported and appear to be critical in the regulation of client protein function. These data indicate that one of the mechanisms by which PTMs contribute to protein function is by facilitating interaction with chaperones. Taken together, we propose that XL-MS analysis of chaperone complexes may be used as a unique way to identify biologically important PTMs on client proteins.

Comprehensive Analysis of G1 Cyclin Docking Motif Sequences that Control CDK Regulatory Potency In Vivo.

Many protein-modifying enzymes recognize their substrates via docking motifs, but the range of functionally permissible motif sequences is often poorly defined. During eukaryotic cell division, cyclin-specific docking motifs help cyclin-dependent kinases (CDKs) phosphorylate different substrates at different stages, thus enforcing a temporally ordered series of events. In budding yeast, CDK substrates with Leu/Pro-rich (LP) docking motifs are recognized by Cln1/2 cyclins in late G1 phase, yet the key sequence features of these motifs were unknown. Here, we comprehensively analyze LP motif requirements in vivo by combining a competitive growth assay with deep mutational scanning. We quantified the effect of all single-residue replacements in five different LP motifs by using six distinct G1 cyclins from diverse fungi including medical and agricultural pathogens. The results uncover substantial tolerance for deviations from the consensus sequence, plus requirements at some positions that are contingent on the favorability of other motif residues. They also reveal the basis for variations in functional potency among wild-type motifs, and allow derivation of a quantitative matrix that predicts the strength of other candidate motif sequences. Finally, we find that variation in docking motif potency can advance or delay the time at which CDK substrate phosphorylation occurs, and thereby control the temporal ordering of cell cycle regulation. The overall results provide a general method for surveying viable docking motif sequences and quantifying their potency in vivo, and they reveal how variations in docking strength can tune the degree and timing of regulatory modifications.

MAPK modulation of yeast pheromone signaling output and the role of phosphorylation sites in the scaffold protein Ste5.

Mitogen-activated protein kinases (MAPKs) mediate numerous eukaryotic signaling responses. They also can modulate their own signaling output via positive or negative feedback loops. In the yeast pheromone response pathway, the MAPK Fus3 triggers negative feedback that dampens its own activity. One target of this feedback is Ste5, a scaffold protein that promotes Fus3 activation. Binding of Fus3 to a docking motif (D motif) in Ste5 causes signal dampening, which was proposed to involve a central cluster of phosphorylation sites in Ste5. Here, we reanalyzed the role of these central sites. Contrary to prior claims, phosphorylation-mimicking mutations at these sites did not impair signaling. Also, the hyperactive signaling previously observed when these sites were mutated to nonphosphorylatable residues arose from their replacement with valine residues and was not observed with other substitutes. Instead, a cluster of N-terminal sites in Ste5, not the central sites, is required for the rapid dampening of initial responses. Further results suggest that the role of the Fus3 D motif is most simply explained by a tethering effect that promotes Ste5 phosphorylation, rather than an allosteric effect proposed to regulate Fus3 activity. These findings substantially revise our understanding of how MAPK feedback attenuates scaffold-mediated signaling in this model pathway.

CDK and MAPK Synergistically Regulate Signaling Dynamics via a Shared Multi-site Phosphorylation Region on the Scaffold Protein Ste5.

We report an unanticipated system of joint regulation by cyclin-dependent kinase (CDK) and mitogen-activated protein kinase (MAPK), involving collaborative multi-site phosphorylation of a single substrate. In budding yeast, the protein Ste5 controls signaling through a G1 arrest pathway. Upon cell-cycle entry, CDK inhibits Ste5 via multiple phosphorylation sites, disrupting its membrane association. Using quantitative time-lapse microscopy, we examined Ste5 membrane recruitment dynamics at different cell-cycle stages. Surprisingly, in S phase, where Ste5 recruitment should be blocked, we observed an initial recruitment followed by a steep drop-off. This delayed inhibition revealed a requirement for both CDK activity and negative feedback from the pathway MAPK Fus3. Mutagenesis, mass spectrometry, and electrophoretic analyses suggest that the CDK and MAPK modify shared sites, which are most extensively phosphorylated when both kinases are active and able to bind their docking sites on Ste5. Such collaborative phosphorylation can broaden regulatory inputs and diversify output dynamics of signaling pathways.

Analysis of the thresholds for transcriptional activation by the yeast MAP kinases Fus3 and Kss1.

Signaling in the pheromone response pathway of budding yeast activates two distinct MAP kinases (MAPKs), Fus3 and Kss1. Either MAPK alone can mediate pheromone-induced transcription, but it has been unclear to what degree each one contributes to transcriptional output in wild-type cells. Here, we report that transcription reflects the ratio of active to inactive MAPK, and not simply the level of active MAPK. For Kss1 the majority of MAPK molecules must be converted to the active form, whereas for Fus3 only a small minority must be activated. These different activation thresholds reflect two opposing effects of each MAPK, in which the inactive forms inhibit transcription, whereas the active forms promote transcription. Moreover, negative feedback from Fus3 limits activation of Kss1 so that it does not meet its required threshold in wild-type cells but does so only when hyperactivated in cells lacking Fus3. The results suggest that the normal transcriptional response involves asymmetric contributions from the two MAPKs, in which pheromone signaling reduces the negative effect of Kss1 while increasing the positive effect of Fus3. These findings reveal new functional distinctions between these MAPKs, and help illuminate how inhibitory functions shape positive pathway outputs in both pheromone and filamentation pathways.

A docking interface in the cyclin Cln2 promotes multi-site phosphorylation of substrates and timely cell-cycle entry.

BACKGROUND: Eukaryotic cell division is driven by cyclin-dependent kinases (CDKs). Distinct cyclin-CDK complexes are specialized to drive different cell-cycle events, though the molecular foundations for these specializations are only partly understood. In budding yeast, the decision to begin a new cell cycle is regulated by three G1 cyclins (Cln1-Cln3). Recent studies revealed that some CDK substrates contain a novel docking motif that is specifically recognized by Cln1 and Cln2, and not by Cln3 or later S- or M-phase cyclins, but the responsible cyclin interface was unknown. RESULTS: Here, to explore the role of this new docking mechanism in the cell cycle, we first show that it is conserved in a distinct cyclin subtype (Ccn1). Then, we exploit phylogenetic variation to identify cyclin mutations that disrupt docking. These mutations disrupt binding to multiple substrates as well as the ability to use docking sites to promote efficient, multi-site phosphorylation of substrates in vitro. In cells where the Cln2 docking function is blocked, we observed reductions in the polarized morphogenesis of daughter buds and reduced ability to fully phosphorylate the G1/S transcriptional repressor Whi5. Furthermore, disruption of Cln2 docking perturbs the coordination between cell size and division, such that the G1/S transition is delayed. CONCLUSIONS: The findings point to a novel substrate interaction interface on cyclins, with patterns of conservation and divergence that relate to functional distinctions among cyclin subtypes. Furthermore, this docking function helps ensure full phosphorylation of substrates with multiple phosphorylation sites, and this contributes to punctual cell-cycle entry.

Regulation of cyclin-substrate docking by a G1 arrest signaling pathway and the Cdk inhibitor Far1.

Eukaryotic cell division is often regulated by extracellular signals. In budding yeast, signaling from mating pheromones arrests the cell cycle in G1 phase. This arrest requires the protein Far1, which is thought to antagonize the G1/S transition by acting as a Cdk inhibitor (CKI), although the mechanisms remain unresolved. Recent studies found that G1/S cyclins (Cln1 and Cln2) recognize Cdk substrates via specific docking motifs, which promote substrate phosphorylation in vivo. Here, we show that these docking interactions are inhibited by pheromone signaling and that this inhibition requires Far1. Moreover, Far1 mutants that cannot inhibit docking are defective at cell-cycle arrest. Consistent with this arrest function, Far1 outcompetes substrates for association with G1/S cyclins in vivo, and it is present in large excess over G1/S cyclins during the precommitment period where pheromone can impose G1 arrest. Finally, a comparison of substrates that do and do not require docking suggests that Far1 acts as a multimode inhibitor that antagonizes both kinase activity and substrate recognition by Cln1/2-Cdk complexes. Our findings uncover a novel mechanism of Cdk regulation by external signals and shed new light on Far1 function to provide a revised view of cell-cycle arrest in this model system.

Functional overlap among distinct G1/S inhibitory pathways allows robust G1 arrest by yeast mating pheromones.

In budding yeast, mating pheromones arrest the cell cycle in G1 phase via a pheromone-activated Cdk-inhibitor (CKI) protein, Far1. Alternate pathways must also exist, however, because deleting the cyclin CLN2 restores pheromone arrest to far1 cells. Here we probe whether these alternate pathways require the G1/S transcriptional repressors Whi5 and Stb1 or the CKI protein Sic1, whose metazoan analogues (Rb or p27) antagonize cell cycle entry. Removing Whi5 and Stb1 allows partial escape from G1 arrest in far1 cln2 cells, along with partial derepression of G1/S genes, which implies a repressor-independent route for inhibiting G1/S transcription. This route likely involves pheromone-induced degradation of Tec1, a transcriptional activator of the cyclin CLN1, because Tec1 stabilization also causes partial G1 escape in far1 cln2 cells, and this is additive with Whi5/Stb1 removal. Deleting SIC1 alone strongly disrupts Far1-independent G1 arrest, revealing that inhibition of B-type cyclin-Cdk activity can empower weak arrest pathways. Of interest, although far1 cln2 sic1 cells escaped G1 arrest, they lost viability during pheromone exposure, indicating that G1 exit is deleterious if the arrest signal remains active. Overall our findings illustrate how multiple distinct G1/S-braking mechanisms help to prevent premature cell cycle commitment and ensure a robust signal-induced G1 arrest.

Cyclin-specific docking motifs promote phosphorylation of yeast signaling proteins by G1/S Cdk complexes.

BACKGROUND: The eukaryotic cell cycle begins with a burst of cyclin-dependent kinase (Cdk) phosphorylation. In budding yeast, several Cdk substrates are preferentially phosphorylated at the G1/S transition rather than later in the cell cycle when Cdk activity levels are high. These early Cdk substrates include signaling proteins in the pheromone response pathway. Two such proteins, Ste5 and Ste20, are phosphorylated only when Cdk is associated with the G1/S cyclins Cln1 and Cln2 and not G1, S, or M cyclins. The basis of this cyclin specificity is unknown. RESULTS: Here we show that Ste5 and Ste20 have recognition sequences, or "docking" sites, for the G1/S cyclins. These docking sites, which are distinct from Clb5/cyclin A-binding "RXL" motifs, bind preferentially to Cln2. They strongly enhance Cln2-driven phosphorylation of each substrate in vivo and function largely independent of position and distance to the Cdk sites. We exploited this functional independence to rewire a Cdk regulatory circuit in a way that changes the target of Cdk inhibition in the pheromone response pathway. Furthermore, we uncover functionally active Cln2 docking motifs in several other Cdk substrates. The docking motifs drive cyclin-specific phosphorylation, and the cyclin preference can be switched by using a distinct motif. CONCLUSIONS: Our findings indicate that some Cdk substrates are intrinsically capable of being phosphorylated by several different cyclin-Cdk forms, but they are inefficiently phosphorylated in vivo without a cyclin-specific docking site. Docking interactions may play a prevalent but previously unappreciated role in driving phosphorylation of select Cdk substrates preferentially at the G1/S transition.

Designing new cellular signaling pathways.

All cells respond to signals from the environment. Extracellular stimuli activate intracellular signal transduction pathways that make decisions about cell identity, behavior, and survival. A nascent field aims to design and construct new signaling pathways beyond those found in nature. Current strategies exploit the structural modularity of many signaling proteins, which makes them inherently amenable to domain-swapping tactics that exchange their input and output connections. The results reveal a remarkable degree of functional plasticity in signaling proteins and pathways, as well as regulatory logic that can be transported to new proteins. Modified adaptor and scaffold proteins can reroute signal traffic and adjust the response behavior of the pathway circuit. These synthetic biology approaches promise to deepen our understanding of existing signaling pathways and spur the development of new cellular tools for research, industry, and medicine.

Membrane localization of scaffold proteins promotes graded signaling in the yeast MAP kinase cascade.

BACKGROUND: Signaling through mitogen-activated protein kinase (MAPK) cascade pathways can show various input-output behaviors, including either switch-like or graded responses to increasing levels of stimulus. Prior studies suggest that switch-like behavior is promoted by positive feedback loops and nonprocessive phosphorylation reactions, but it is unclear whether graded signaling is a default behavior or whether it must be enforced by separate mechanisms. It has been hypothesized that scaffold proteins promote graded behavior. RESULTS: Here, we experimentally probe the determinants of graded signaling in the yeast mating MAPK pathway. We find that graded behavior is robust in that it resists perturbation by loss of several negative-feedback regulators. However, the pathway becomes switch-like when activated by a crosstalk stimulus that bypasses multiple upstream components. To dissect the contributing factors, we developed a method for gradually varying the signal input at different pathway steps in vivo. Input at the beginning of the kinase cascade produced a sharp, threshold-like response. Surprisingly, the scaffold protein Ste5 increased this threshold behavior when limited to the cytosol. However, signaling remained graded whenever Ste5 was allowed to function at the plasma membrane. CONCLUSIONS: The results suggest that the MAPK cascade module is inherently ultrasensitive but is converted to a graded system by the pathway-specific activation mechanism. Scaffold-mediated assembly of signaling complexes at the plasma membrane allows faithful propagation of weak signals, which consequently reduces pathway ultrasensitivity. These properties help shape the input-output properties of the system to fit the physiological context.

Distinct roles for two Galpha-Gbeta interfaces in cell polarity control by a yeast heterotrimeric G protein.

Saccharomyces cerevisiae mating pheromones trigger dissociation of a heterotrimeric G protein (Galphabetagamma) into Galpha-guanosine triphosphate (GTP) and Gbetagamma. The Gbetagamma dimer regulates both mitogen-activated protein (MAP) kinase cascade signaling and cell polarization. Here, by independently activating the MAP kinase pathway, we studied the polarity role of Gbetagamma in isolation from its signaling role. MAP kinase signaling alone could induce cell asymmetry but not directional growth. Surprisingly, active Gbetagamma, either alone or with Galpha-GTP, could not organize a persistent polarization axis. Instead, following pheromone gradients (chemotropism) or directional growth without pheromone gradients (de novo polarization) required an intact receptor-Galphabetagamma module and GTP hydrolysis by Galpha. Our results indicate that chemoattractant-induced cell polarization requires continuous receptor-Galphabetagamma communication but not modulation of MAP kinase signaling. To explore regulation of Gbetagamma by Galpha, we mutated Gbeta residues in two structurally distinct Galpha-Gbeta binding interfaces. Polarity control was disrupted only by mutations in the N-terminal interface, and not the Switch interface. Incorporation of these mutations into a Gbeta-Galpha fusion protein, which enforces subunit proximity, revealed that Switch interface dissociation regulates signaling, whereas the N-terminal interface may govern receptor-Galphabetagamma coupling. These findings raise the possibility that the Galphabetagamma heterotrimer can function in a partially dissociated state, tethered by the N-terminal interface.

Identification of novel membrane-binding domains in multiple yeast Cdc42 effectors.

The Rho-type GTPase Cdc42 is a central regulator of eukaryotic cell polarity and signal transduction. In budding yeast, Cdc42 regulates polarity and mitogen-activated protein (MAP) kinase signaling in part through the PAK-family kinase Ste20. Activation of Ste20 requires a Cdc42/Rac interactive binding (CRIB) domain, which mediates its recruitment to membrane-associated Cdc42. Here, we identify a separate domain in Ste20 that interacts directly with membrane phospholipids and is critical for its function. This short region, termed the basic-rich (BR) domain, can target green fluorescent protein to the plasma membrane in vivo and binds PIP(2)-containing liposomes in vitro. Mutation of basic or hydrophobic residues in the BR domain abolishes polarized localization of Ste20 and its function in both MAP kinase-dependent and independent pathways. Thus, Cdc42 binding is required but is insufficient; instead, direct membrane binding by Ste20 is also required. Nevertheless, phospholipid specificity is not essential in vivo, because the BR domain can be replaced with several heterologous lipid-binding domains of varying lipid preferences. We also identify functionally important BR domains in two other yeast Cdc42 effectors, Gic1 and Gic2, suggesting that cooperation between protein-protein and protein-membrane interactions is a prevalent mechanism during Cdc42-regulated signaling and perhaps for other dynamic localization events at the cell cortex.

A mechanism for cell-cycle regulation of MAP kinase signaling in a yeast differentiation pathway.

Yeast cells arrest in the G1 phase of the cell cycle upon exposure to mating pheromones. As cells commit to a new cycle, G1 CDK activity (Cln/CDK) inhibits signaling through the mating MAPK cascade. Here we show that the target of this inhibition is Ste5, the MAPK cascade scaffold protein. Cln/CDK disrupts Ste5 membrane localization by phosphorylating a cluster of sites that flank a small, basic, membrane-binding motif in Ste5. Effective inhibition of Ste5 signaling requires multiple phosphorylation sites and a substantial accumulation of negative charge, which suggests that Ste5 acts as a sensor for high G1 CDK activity. Thus, Ste5 is an integration point for both external and internal signals. When Ste5 cannot be phosphorylated, pheromone triggers an aberrant arrest of cells outside G1 either in the presence or absence of the CDK-inhibitor protein Far1. These findings define a mechanism and physiological benefit of restricting antiproliferative signaling to G1.

Dual role for membrane localization in yeast MAP kinase cascade activation and its contribution to signaling fidelity.

Distinct MAP kinase pathways in yeast share several signaling components , including the PAK Ste20 and the MAPKKK Ste11, yet signaling is specific. Mating pheromones trigger an initial step in which Ste20 activates Ste11 , and this requires plasma membrane recruitment of the MAP kinase cascade scaffold protein, Ste5 . Here, we demonstrate an additional role for Ste5 membrane localization. Once Ste11 is activated, signaling through the mating pathway remains minimal but is substantially amplified when Ste5 is recruited to the membrane either by the Gbetagamma dimer or by direct membrane targeting, even to internal membranes. Ste11 signaling is also amplified by Ste5 oligomerization and by a hyperactivating mutation in the Ste7 binding region of Ste5. We suggest a model in which membrane recruitment of Ste5 concentrates its binding partners and thereby amplifies signaling through the kinase cascade. We find similar behavior in the osmotically responsive HOG pathway. Remarkably, while both pheromone and hyperosmotic stimuli amplify signaling from constitutively active Ste11, the resulting signaling output remains pathway specific. These findings suggest a common mode of regulation in which pathway stimuli both initiate and amplify MAP kinase cascade signaling. The regulation of rate-limiting steps that lie after a branchpoint from shared components helps ensure signaling specificity.

A membrane binding domain in the ste5 scaffold synergizes with gbetagamma binding to control localization and signaling in pheromone response.

Activation of mitogen-activated protein (MAP) kinase cascade signaling by yeast mating pheromones involves recruitment of the Ste5 scaffold protein to the plasma membrane by the receptor-activated Gbetagamma dimer. Here, we identify a putative amphipathic alpha-helical domain in Ste5 that binds directly to phospholipid membranes and is required for membrane recruitment by Gbetagamma. Thus, Ste5 signaling requires synergistic Ste5-Gbetagamma and Ste5-membrane interactions, with neither alone being sufficient. Remarkably, the Ste5 membrane binding domain is a dual-function motif that also mediates nuclear import. Separation-of-function mutations show that signaling requires the membrane-targeting activity of this domain, not its nuclear-targeting activity, and heterologous lipid binding domains can substitute for its function. This domain also contains imperfections that reduce membrane affinity, and their elimination results in constitutive signaling, explaining some previous hyperactive Ste5 mutants. Therefore, weak membrane affinity is advantageous, ensuring a normal level of signaling quiescence in the absence of stimulus and imposing a requirement for Gbetagamma binding.

Interaction with the SH3 domain protein Bem1 regulates signaling by the Saccharomyces cerevisiae p21-activated kinase Ste20.

The Saccharomyces cerevisiae PAK (p21-activated kinase) family kinase Ste20 functions in several signal transduction pathways, including pheromone response, filamentous growth, and hyperosmotic resistance. The GTPase Cdc42 localizes and activates Ste20 by binding to an autoinhibitory motif within Ste20 called the CRIB domain. Another factor that functions with Ste20 and Cdc42 is the protein Bem1. Bem1 has two SH3 domains, but target ligands for these domains have not been described. Here we identify an evolutionarily conserved binding site for Bem1 between the CRIB and kinase domains of Ste20. Mutation of tandem proline-rich (PxxP) motifs in this region disrupts Bem1 binding, suggesting that it serves as a ligand for a Bem1 SH3 domain. These PxxP motif mutations affect signaling additively with CRIB domain mutations, indicating that Bem1 and Cdc42 make separable contributions to Ste20 function, which cooperate to promote optimal signaling. This PxxP region also binds another SH3 domain protein, Nbp2, but analysis of bem1Delta versus nbp2Delta strains shows that the signaling defects of PxxP mutants result from impaired binding to Bem1 rather than from impaired binding to Nbp2. Finally, the PxxP mutations also reduce signaling by constitutively active Ste20, suggesting that postactivation functions of PAKs can be promoted by SH3 domain proteins, possibly by colocalizing PAKs with their substrates. The overall results also illustrate how the final signaling function of a protein can be governed by combinatorial addition of multiple, independent protein-protein interaction modules.

Cdc42 regulation of kinase activity and signaling by the yeast p21-activated kinase Ste20.

The Saccharomyces cerevisiae kinase Ste20 is a member of the p21-activated kinase (PAK) family with several functions, including pheromone-responsive signal transduction. While PAKs are usually activated by small G proteins and Ste20 binds Cdc42, the role of Cdc42-Ste20 binding has been controversial, largely because Ste20 lacking its entire Cdc42-binding (CRIB) domain retains kinase activity and pheromone response. Here we show that, unlike CRIB deletion, point mutations in the Ste20 CRIB domain that disrupt Cdc42 binding also disrupt pheromone signaling. We also found that Ste20 kinase activity is stimulated by GTP-bound Cdc42 in vivo and this effect is blocked by the CRIB point mutations. Moreover, the Ste20 CRIB and kinase domains bind each other, and mutations that disrupt this interaction cause hyperactive kinase activity and bypass the requirement for Cdc42 binding. These observations demonstrate that the Ste20 CRIB domain is autoinhibitory and that this negative effect is antagonized by Cdc42 to promote Ste20 kinase activity and signaling. Parallel results were observed for filamentation pathway signaling, suggesting that the requirement for Cdc42-Ste20 interaction is not qualitatively different between the mating and filamentation pathways. While necessary for pheromone signaling, the role of the Cdc42-Ste20 interaction does not require regulation by pheromone or the pheromone-activated G beta gamma complex, because the CRIB point mutations also disrupt signaling by activated forms of the kinase cascade scaffold protein Ste5. In total, our observations indicate that Cdc42 converts Ste20 to an active form, while pathway stimuli regulate the ability of this active Ste20 to trigger signaling through a particular pathway.