Brian J. Yeh

Rewiring cellular morphology pathways with synthetic guanine nucleotide exchange factors

Eukaryotic cells mobilize the actin cytoskeleton to generate a remarkable diversity of morphological behaviours, including motility, phagocytosis and cytokinesis. Much of this diversity is mediated by guanine nucleotide exchange factors (GEFs) that activate Rho family GTPases—the master regulators of the actin cytoskeleton. There are over 80 Rho GEFs in the human genome (compared to only 22 genes for the Rho GTPases themselves), and the evolution of new and diverse GEFs is thought to provide a mechanism for linking the core cytoskeletal machinery to a wide range of new control inputs. Here we test this hypothesis and ask if we can systematically reprogramme cellular morphology by engineering synthetic GEF proteins. We focused on Dbl family Rho GEFs, which have a highly modular structure common to many signalling proteins: they contain a catalytic Dbl homology (DH) domain linked to diverse regulatory domains, many of which autoinhibit GEF activity. Here we show that by recombining catalytic GEF domains with new regulatory modules, we can generate synthetic GEFs that are activated by non-native inputs. We have used these synthetic GEFs to reprogramme cellular behaviour in diverse ways. The GEFs can be used to link specific cytoskeletal responses to normally unrelated upstream signalling pathways. In addition, multiple synthetic GEFs can be linked as components in series to form an artificial cascade with improved signal processing behaviour. These results show the high degree of evolutionary plasticity of this important family of modular signalling proteins, and indicate that it may be possible to use synthetic biology approaches to manipulate the complex spatio-temporal control of cell morphology.

Synthetic biology: lessons from the history of synthetic organic chemistry

The mid-nineteenth century saw the development of a radical new direction in chemistry: instead of simply analyzing existing molecules, chemists began to synthesize them—including molecules that did not exist in nature. The combination of this new synthetic approach with more traditional analytical approaches revolutionized chemistry, leading to a deep understanding of the fundamental principles of chemical structure and reactivity and to the emergence of the modern pharmaceutical and chemical industries. The history of synthetic chemistry offers a possible roadmap for the development and impact of synthetic biology, a nascent field in which the goal is to build novel biological systems.

The NDR/LATS family kinase Cbk1 directly controls transcriptional asymmetry.

Cell fate can be determined by asymmetric segregation of gene expression regulators. In the budding yeast Saccharomyces cerevisiae, the transcription factor Ace2 accumulates specifically in the daughter cell nucleus, where it drives transcription of genes that are not expressed in the mother cell. The NDR/LATS family protein kinase Cbk1 is required for Ace2 segregation and function. Using peptide scanning arrays, we determined Cbk1′s phosphorylation consensus motif, the first such unbiased approach for an enzyme of this family, showing that it is a basophilic kinase with an unusual preference for histidine −5 to the phosphorylation site. We found that Cbk1 phosphorylates such sites in Ace2, and that these modifications are critical for Ace2′s partitioning and function. Using proteins marked with GFP variants, we found that Ace2 moves from isotropic distribution to the daughter cell nuclear localization, well before cytokinesis, and that the nucleus must enter the daughter cell for Ace2 accumulation to occur. We found that Cbk1, unlike Ace2, is restricted to the daughter cell. Using both in vivo and in vitro assays, we found that two critical Cbk1 phosphorylations block Ace2′s interaction with nuclear export machinery, while a third distal modification most likely acts to increase the transcription factors activity. Our findings show that Cbk1 directly controls Ace2, regulating the transcription factors activity and interaction with nuclear export machinery through three phosphorylation sites. Furthermore, Cbk1 exhibits a novel specificity that is likely conserved among related kinases from yeast to metazoans. Cbk1 is functionally restricted to the daughter cell, and cannot diffuse from the daughter to the mother. In addition to providing a mechanism for Ace2 segregation, these findings show that an isotropically distributed cell fate determinant can be asymmetrically partitioned in cytoplasmically contiguous cells through spatial segregation of a regulating protein kinase.

Proteome-wide discovery of evolutionary conserved sequences in disordered regions.

A statistical analysis method can identify short, functionally important linear motifs in disordered regions of proteins. Finding the Hidden Meaning in Disordered Regions Many proteins, including those involved in signal transduction, have large disordered regions, in addition to their clearly defined domains or motifs. Although these disordered regions are functionally important, identifying the important residues in these regions has proved challenging because the regions are not visualized in crystal structures and tend to exhibit high sequence divergence. Nguyen Ba et al. modified the phylogenetic hidden Markov model so that it could be applied to these disordered regions. Application of this method to yeast proteins not only revealed the presence of known short conserved motifs in proteins not known to have these motifs but also predicted previously unknown short conserved motifs. Experimental analysis suggested that both sets of motifs were functionally important. Thus, this approach should provide an effective method for discovering biologically important conserved motifs within the disordered regions of proteins. At least 30% of human proteins are thought to contain intrinsically disordered regions, which lack stable structural conformation. Despite lacking enzymatic functions and having few protein domains, disordered regions are functionally important for protein regulation and contain short linear motifs (short peptide sequences involved in protein-protein interactions), but in most disordered regions, the functional amino acid residues remain unknown. We searched for evolutionarily conserved sequences within disordered regions according to the hypothesis that conservation would indicate functional residues. Using a phylogenetic hidden Markov model (phylo-HMM), we made accurate, specific predictions of functional elements in disordered regions even when these elements are only two or three amino acids long. Among the conserved sequences that we identified were previously known and newly identified short linear motifs, and we experimentally verified key examples, including a motif that may mediate interaction between protein kinase Cbk1 and its substrates. We also observed that hub proteins, which interact with many partners in a protein interaction network, are highly enriched in these conserved sequences. Our analysis enabled the systematic identification of the functional residues in disordered regions and suggested that at least 5% of amino acids in disordered regions are important for function.

Control of protein signaling using a computationally designed GTPase/GEF orthogonal pair

Signaling pathways depend on regulatory protein-protein interactions; controlling these interactions in cells has important applications for reengineering biological functions. As many regulatory proteins are modular, considerable progress in engineering signaling circuits has been made by recombining commonly occurring domains. Our ability to predictably engineer cellular functions, however, is constrained by complex crosstalk observed in naturally occurring domains. Here we demonstrate a strategy for improving and simplifying protein network engineering: using computational design to create orthogonal (non-crossreacting) protein-protein interfaces. We validated the design of the interface between a key signaling protein, the GTPase Cdc42, and its activator, Intersectin, biochemically and by solving the crystal structure of the engineered complex. The designed GTPase (orthoCdc42) is activated exclusively by its engineered cognate partner (orthoIntersectin), but maintains the ability to interface with other GTPase signaling circuit components in vitro. In mammalian cells, orthoCdc42 activity can be regulated by orthoIntersectin, but not wild-type Intersectin, showing that the designed interaction can trigger complex processes. Computational design of protein interfaces thus promises to provide specific components that facilitate the predictable engineering of cellular functions.

The Structure of an NDR/LATS Kinase-Mob Complex Reveals a Novel Kinase-Coactivator System and Substrate Docking Mechanism.

Eukaryotic cells commonly use protein kinases in signaling systems that relay information and control a wide range of processes. These enzymes have a fundamentally similar structure, but achieve functional diversity through variable regions that determine how the catalytic core is activated and recruited to phosphorylation targets. “Hippo” pathways are ancient protein kinase signaling systems that control cell proliferation and morphogenesis; the NDR/LATS family protein kinases, which associate with “Mob” coactivator proteins, are central but incompletely understood components of these pathways. Here we describe the crystal structure of budding yeast Cbk1–Mob2, to our knowledge the first of an NDR/LATS kinase–Mob complex. It shows a novel coactivator-organized activation region that may be unique to NDR/LATS kinases, in which a key regulatory motif apparently shifts from an inactive binding mode to an active one upon phosphorylation. We also provide a structural basis for a substrate docking mechanism previously unknown in AGC family kinases, and show that docking interaction provides robustness to Cbk1’s regulation of its two known in vivo substrates. Co-evolution of docking motifs and phosphorylation consensus sites strongly indicates that a protein is an in vivo regulatory target of this hippo pathway, and predicts a new group of high-confidence Cbk1 substrates that function at sites of cytokinesis and cell growth. Moreover, docking peptides arise in unstructured regions of proteins that are probably already kinase substrates, suggesting a broad sequential model for adaptive acquisition of kinase docking in rapidly evolving intrinsically disordered polypeptides.

Reengineering the signaling properties of a Src family kinase.

Src family kinases (SFKs) are modular signaling proteins possessing SH3, SH2, and tyrosine kinase domains. The SH3 and SH2 domains of SFKs have dual roles: they regulate the activity of the kinases, and they also target SFKs to their cellular substrates. We generated a series of novel SFKs by replacing the SH2 and SH3 domains of Hck with the syntrophin PDZ domain. In some constructs, the negative regulatory tyrosine in the C-terminal tail was also replaced with a PDZ ligand sequence. When expressed in mammalian cells, the substrate specificity of the PDZ-kinases was directed to a different group of proteins than wild-type Hck. The PDZ-kinases phosphorylate neuronal nitric oxide synthase (nNOS), a known binding partner of the syntrophin PDZ domain. We also introduced a PDZ ligand at the C-terminus of the adaptor protein Cas. PDZ-Hck kinases phosphorylate the engineered Cas protein in Cas(-/-) cells and restore the migration defect of these cells. A PDZ-kinase was also functional in rewiring MAPK signaling via an engineered ErbB2 construct containing a PDZ ligand sequence. Several of the PDZ-kinases show autoregulatory properties similar to natural SFKs. Thus, the PDZ-ligand interaction is able to functionally replace the normal SH2-pY527 interaction that regulates SFKs. Our data highlight the modularity and evolvability of signaling proteins.