Sarah Cox

Domain movements in protein kinases

Structural studies of the catalytic subunit of the cAMP-dependent protein kinase, both by crystallographic methods and in solution, reveal two conformations. Crystal structures of several other protein kinases have also been solved in the past year. With this combined information we can begin to define mobile domains and subdomains within the conserved catalytic core.

Invariance of Initiation Mass and Predictability of Cell Size in Escherichia coli

It is generally assumed that the allocation and synthesis of total cellular resources in microorganisms are uniquely determined by the growth conditions. Adaptation to a new physiological state leads to a change in cell size via reallocation of cellular resources. However, it has not been understood how cell size is coordinated with biosynthesis and robustly adapts to physiological states. We show that cell size in Escherichia coli can be predicted for any steady-state condition by projecting all biosynthesis into three measurable variables representing replication initiation, replication-division cycle, and the global biosynthesis rate. These variables can be decoupled by selectively controlling their respective core biosynthesis using CRISPR interference and antibiotics, verifying our predictions that different physiological states can result in the same cell size. We performed extensive growth inhibition experiments, and we discovered that cell size at replication initiation per origin, namely the initiation mass or unit cell, is remarkably invariant under perturbations targeting transcription, translation, ribosome content, replication kinetics, fatty acid and cell wall synthesis, cell division, and cell shape. Based on this invariance and balanced resource allocation, we explain why the total cell size is the sum of all unit cells. These results provide an overarching framework with quantitative predictive power over cell size in bacteria.

Sbf/MTMR13 coordinates PI(3)P and Rab21 regulation in endocytic control of cellular remodeling.

The MTM phosphatases include poorly defined, catalytically inactive members. Drosophila Sbf, an MTM pseudophosphatase, physically and functionally interacts with class II PI3-kinase, Mtm PI3-phosphatase, and Rab21, each required for macrophage remodeling. Sbf plays dual roles in Mtm PI(3)P turnover and as a Rab21 GEF to coordinate endosomal dynamics.

Starvation-induced MTMR13 and RAB21 activity regulates VAMP8 to promote autophagosome- lysosome fusion

Autophagy, the process for recycling cytoplasm in the lysosome, depends on membrane trafficking. We previously identified Drosophila Sbf as a Rab21 guanine nucleotide exchange factor (GEF) that acts with Rab21 in endosomal trafficking. Here, we show that Sbf/MTMR13 and Rab21 have conserved functions required for starvation‐induced autophagy. Depletion of Sbf/MTMR13 or Rab21 blocked endolysosomal trafficking of VAMP8, a SNARE required for autophagosome–lysosome fusion. We show that starvation induces Sbf/MTMR13 GEF and RAB21 activity, as well as their induced binding to VAMP8 (or closest Drosophila homolog, Vamp7). MTMR13 is required for RAB21 activation, VAMP8 interaction and VAMP8 endolysosomal trafficking, defining a novel GEF‐Rab‐effector pathway. These results identify starvation‐responsive endosomal regulators and trafficking that tunes membrane demands with changing autophagy status.

Substrate recognition and ubiquitination of SCFSkp2/Cks1 ubiquitin-protein isopeptide ligase

p27, an important cell cycle regulator, blocks the G1/S transition in cells by binding and inhibiting Cdk2/cyclin A and Cdk2/cyclin E complexes (Cdk2/E). Ubiquitination and subsequent degradation play a critical role in regulating the levels of p27 during cell cycle progression. Here we provide evidence suggesting that both Cdk2/E and phosphorylation of Thr187 on p27 are essential for the recognition of p27 by the SCFSkp2/Cks1 complex, the ubiquitin-protein isopeptide ligase (E3). Cdk2/E provides a high affinity binding site, whereas the phosphorylated Thr187 provides a low affinity binding site for the Skp2/Cks1 complex. Furthermore, binding of phosphorylated p27/Cdk2/E to the E3 complex showed positive cooperativity. Consistently, p27 is also ubiquitinated in a similarly cooperative manner. In the absence of p27, Cdk2/E and Cks1 increase Skp2 phosphorylation. This phosphorylation enhances Skp2 auto-ubiquitination, whereas p27 inhibits both phosphorylation and auto-ubiquitination of Skp2.

In Vitro SCFβ‐Trcp1–Mediated IκBα Ubiquitination Assay for High‐Throughput Screen

An increasing body of evidence indicates that constitutive activation of NF-kappaB contributes to tumorigenesis and inflammation. Ubiquitination and degradation of IkappaB plays an essential role in NF-kappaB activation. Here we describe an in vitro IkappaBalpha ubiquitination assay system in which purified E1, E2, SCF(beta-Trcp1) E3, IkappaBalpha, IKK2, and Ub were used to generate ubiquitinated IkappaBalpha. The ubiquitination of IkappaBalpha is strictly dependent on its phosphorylation by IKK2, as well as the presence of E1, E2, E3, and Ub. The assay was adapted into 384-well plate format in which an antibody against IkappaBalpha was used to capture IkappaBalpha, and the biotinylated ubiquitin attached to IkappaBalpha was detected with europium (Eu)-labeled streptavidin. This assay can be used to discover inhibitors of IkappaBalpha ubiquitination. Such inhibitors would block NF-kappaB activation by stabilizing IkappaB levels in cells and thus provide a new therapeutic approach to NF-kappaB-related human diseases.

Deconstructing cell size control into physiological modules in Escherichia coli

It is generally assumed that the allocation and synthesis of total cellular resources in microorganisms are uniquely determined by the growth conditions. Adaptation to a new physiological state leads to a change in cell size via reallocation of cellular resources. However, it has not been understood how cell size is coordinated with biosynthesis and robustly adapts to physiological states. We show that cell size in Escherichia coli can be predicted for any steady-state condition by projecting all biosynthesis into three measurable variables representing replication initiation, replication-division cycle, and the global biosynthesis rate. These variables can be decoupled by selectively controlling their respective core biosynthesis using CRISPR interference and antibiotics, verifying our predictions that different physiological states can result in the same cell size. We performed extensive growth inhibition experiments, and discovered that cell size at replication initiation per origin, namely the initiation mass or “unit cell,” is remarkably invariant under perturbations targeting transcription, translation, ribosome content, replication kinetics, fatty acid and cell-wall synthesis, cell division, and cell shape. Based on this invariance and balanced resource allocation, we explain why the total cell size is the sum of all unit cells. These results provide an overarching framework with quantitative predictive power over cell size in bacteria.