Chuan‐Hsiang Huang

PIK3CA , one of the two most frequently mutated oncogenes in human tumors, codes for p110α, the catalytic subunit of a phosphatidylinositol 3-kinase, isoform α (PI3Kα, p110α/p85). Here, we report a 3.0 angstrom resolution structure of a complex between p110α and a polypeptide containing the p110α-binding domains of p85α, a protein required for its enzymatic activity. The structure shows that many of the mutations occur at residues lying at the interfaces between p110α and p85α or between the kinase domain of p110α and other domains within the catalytic subunit. Disruptions of these interactions are likely to affect the regulation of kinase activity by p85 or the catalytic activity of the enzyme, respectively. In addition to providing new insights about the structure of PI3Kα, these results suggest specific mechanisms for the effect of oncogenic mutations in p110α and p85α.

Chemotaxis, the directed migration of cells in chemical gradients, is a vital process in normal physiology and in the pathogenesis of many diseases. Chemotactic cells display motility, directional sensing, and polarity. Motility refers to the random extension of pseudopodia, which may be driven by spontaneous actin waves that propagate through the cytoskeleton. Directional sensing is mediated by a system that detects temporal and spatial stimuli and biases motility toward the gradient. Polarity gives cells morphologically and functionally distinct leading and lagging edges by relocating proteins or their activities selectively to the poles. By exploiting the genetic advantages of Dictyostelium, investigators are working out the complex network of interactions between the proteins that have been implicated in the chemotactic processes of motility, directional sensing, and polarity.

Mutations in oncogenes often promote tumorigenesis by changing the conformation of the encoded proteins, thereby altering enzymatic activity. The PIK3CA oncogene, which encodes p110alpha, the catalytic subunit of phosphatidylinositol 3-kinase alpha (PI3Kalpha), is one of the two most frequently mutated oncogenes in human cancers. We report the structure of the most common mutant of p110alpha in complex with two interacting domains of its regulatory partner (p85alpha), both free and bound to an inhibitor (wortmannin). The N-terminal SH2 (nSH2) domain of p85alpha is shown to form a scaffold for the entire enzyme complex, strategically positioned to communicate extrinsic signals from phosphopeptides to three distinct regions of p110alpha. Moreover, we found that Arg-1047 points toward the cell membrane, perpendicular to the orientation of His-1047 in the WT enzyme. Surprisingly, two loops of the kinase domain that contact the cell membrane shift conformation in the oncogenic mutant. Biochemical assays revealed that the enzymatic activity of the p110alpha His1047Arg mutant is differentially regulated by lipid membrane composition. These structural and biochemical data suggest a previously undescribed mechanism for mutational activation of a kinase that involves perturbation of its interaction with the cellular membrane.

Cells have an internal compass that enables them to move along shallow chemical gradients. As amoeboid cells migrate, signaling events such as Ras and PI3K activation occur spontaneously on pseudopodia. Uniform stimuli trigger a symmetric response, whereupon cells stop and round up; then localized patches of activity appear as cells spread. Finally cells adapt and resume random migration. In contrast, chemotactic gradients continuously direct signaling events to the front of the cell. Local-excitation, global-inhibition (LEGI) and reaction-diffusion models have captured some of these features of chemotaxing cells, but no system has explained the complex response kinetics, sensitivity to shallow gradients, or the role of recently observed propagating waves within the actin cytoskeleton. We report here that Ras and PI3K activation move in phase with the cytoskeleton events and, drawing on all of these observations, propose the LEGI-biased excitable network hypothesis. We formulate a model that simulates most of the behaviors of chemotactic cells: In the absence of stimulation, there are spontaneous spots of activity. Stimulus increments trigger an initial burst of patches followed by localized secondary events. After a few minutes, the system adapts, again displaying random activity. In gradients, the activity patches are directed continuously and selectively toward the chemoattractant, providing an extraordinary degree of amplification. Importantly, by perturbing model parameters, we generate distinct behaviors consistent with known classes of mutants. Our study brings together heretofore diverse observations on spontaneous cytoskeletal activity, signaling responses to temporal stimuli, and spatial gradient sensing into a unified scheme.