Our major focus in the area of cell signaling is the allosteric control of protein kinases, which are crucial for cellular signal transduction because of their ability to phosphorylate proteins. We seek to define the mechanisms that keep these kinases inactive until a signal arrives, and to understand how the signal converts the kinase to an active form. Since the aberrant activation of kinases underlies many cancers, our work has relevance for understanding how cancer drugs work, and how they might be improved.
Activation of the epidermal growth factor receptor. Receptor tyrosine kinases span the cell membrane and coordinate the first step in transmitting extracellular signals to the cellular machinery. We study the epidermal growth factor receptor (EGFR, also known as ErbB1 or Her1) and its three close relatives: ErbB2/Her2 and ErbB4/Her4, which are kinase-active, and ErbB3/Her3, which is not.
We discovered that the activation of the catalytic domain of EGFR family members is controlled primarily by an allosteric interaction between two protein kinase domains in an asymmetric dimer, rather than by phosphorylation. In this mechanism, which contrasts with the phosphorylation-based activation mechanism of most receptor tyrosine kinases, the kinase domain of one receptor molecule plays a role analogous to that of a cyclin bound to a cyclin-dependent protein kinase, and induces a conformational change in the kinase domain of a second receptor. We demonstrated that the segments of the receptor that connect the transmembrane segments to the kinase domains, known as the juxtamembrane segments, latch the kinase domains together so as to stabilize the activating asymmetric kinase domain dimer. Part of the interaction involves short α-helices within the juxtamembrane segment that are likely to interact in an antiparallel manner, such that they connect readily to the ends of the dimeric form of the transmembrane helices. Our data are consistent with a model in which the cytoplasmic domains of the receptor are capable of dimerizing and activating on their own. The extracellular domains make the activation ligand-dependent by blocking the activating dimerization in the absence of ligand.
The inhibition of the Abl tyrosine kinase by imatinib. We had discovered some time ago that the recognition of an inactive conformation of Abl, in which a catalytically important Asp-Phe-Gly (DFG) motif at the heart of the kinase domain is flipped by ~180o with respect to the active conformation, underlies the specificity of the cancer drug imatinib (Gleevec). Imatinib binds to the c-Src tyrosine kinase about 10,000-fold less tightly than to Abl, which is puzzling because every residue that makes contact with the drug in Abl is either the same or replaced with a similar residue in c-Src. We have recently worked out why Gleevec binds to Abl but not to Src. The DFG motif can be flipped readily in both c-Src and Abl, but differences in a loop that covers the ATP molecule (known as the P-loop) weakens the binding of Gleevec to Abl but not to c-Src. The P-loop is the site of several important resistance mutations in cancer patients taking imatinib, consistent with its role in stabilizing the binding of the drug. This work involves collaborations with Neil Shah (University of California, San Francisco), Kevan Shokat (HHMI, UCSF), and David Shaw (D. E. Shaw Research).
The recognition of c-Src by its inactivator Csk. The catalytic activity of the Src family of tyrosine kinases is suppressed by phosphorylation on a tyrosine residue located near the C terminus (Tyr 527 in c-Src), which is catalyzed by C-terminal Src kinase (Csk). Given the promiscuity of most tyrosine kinases, it is remarkable that the C-terminal tails of the Src family kinases are the only known targets of Csk. We found that interactions between these kinases position the C-terminal tail of c-Src at the edge of the active site of Csk. Csk cannot phosphorylate substrates that lack this docking mechanism because the conventional substrate-binding site used by most tyrosine kinases to recognize substrates is destabilized in Csk by a deletion in the activation loop.
Autoregulation of ZAP-70. ZAP-70, a cytoplasmic tyrosine kinase required for T cell antigen receptor signaling, is controlled by a regulatory segment that includes a tandem SH2 (Src-homology 2) unit responsible for binding to closely spaced phosphotyrosine residues in the tails of proteins associated with the T cell receptor. In collaboration with Arthur Weiss (HHMI, UCSF), we have worked out how the two SH2 domains in ZAP-70 keep the kinase activity suppressed. When not bound to their phosphotyrosine targets, the SH2 domains engage the hinge region of kinase domain, reducing flexibility. Binding to phosphotyrosine, or phosphorylation of a linker segment, releases the SH2 domains and activates the kinase. We now seek to understand how the ZAP-70 mechanism is integrated into the signal transduction cascade originating at the T cell receptor.
Activation of Ras by nucleotide exchange. Ras is a small membrane-bound guanine nucleotidebinding protein that transmits signals when bound to GTP. The activation of tyrosine kinase receptors results in the recruitment of the Ras-specific nucleotide exchange factor SOS to the membrane, where it encounters Ras and catalyzes the exchange of bound GDP for GTP. Our serendipitous discovery that Ras, which is the substrate of SOS, is itself required for SOS activity is helping us understand a complex mechanism that regulates the activation of Ras by SOS. Ras·GTP is a potent mediator of cell behavior, morphology, and fate, and the Ras activation mechanism that we are uncovering appears to minimize the risk of accidental activation of Ras.
A key advance occurred by studying the activation of Ras localized either to small unilamellar vesicles or to supported lipid bilayers (previous work was carried out with soluble forms of Ras and SOS). We discovered that the second Ras-binding site in SOS can recruit SOS to membranes via Ras, and that this increases the activity of SOS by as much as 500-fold, due to the enhanced encounter with substrate Ras on membranes. Much of the regulatory mechanism concerns control of access to this second site, with an important role played by membrane components such as the inositol lipid PIP2 (phosphatidylinositol-4,5-bisphosphate). This work is carried out in collaboration with Dafna Bar-Sagi (New York University) and Jay Groves (HHMI, UC Berkeley).
Processive DNA Replication
DNA polymerases that replicate chromosomes achieve high speed by utilizing specialized proteins that allow the polymerase to move rapidly along DNA without letting go. These proteins include the "sliding DNA clamp" (the β clamp in Escherichia coli; PCNA [proliferating cell nuclear antigen] in eukaryotes) and the clamp-loader complex (γ complex in E. coli; RFC [replication factor C] complex in eukaryotes) that couples ATP binding and hydrolysis to the opening of the β clamp and its loading onto DNA. Rapid movement of the replication fork involves the coordinated action of two polymerase-exonuclease complexes, working with sliding clamps and a clamp-loader complex.
We work closely with Michael O'Donnell (HHMI, Rockefeller University) to explore the molecular mechanism of high-speed DNA replication. One recent advance in this area is the determination of the structure of a clamp-loader complex bound to a primer-template junction and fully loaded with an ATP analog. This structure explains how recognition of the proper DNA target is coupled to ATP hydrolysis and release of the sliding clamp on DNA.
The work on the epidermal growth factor receptor is supported in part by a grant from the National Cancer Institute. The work on DNA replication is supported in part by a grant from the National Institutes of Health. The work on Abl is supported in part by a grant from the Leukemia and Lymphoma Society.
As of May 30, 2012