My laboratory established in vitro systems to study the biosynthesis, fatty acylation, and membrane insertion of Src kinases. We identified a novel membrane-binding motif within the Src protein consisting of myristate plus a cluster of basic residues. Membrane association results from synergism provided by hydrophobic insertion of myristate into the lipid bilayer and electrostatic interaction of the positively charged amino acids with negatively charged head groups of acidic membrane phospholipids. This “myristate + basic” motif is also found in the Gag proteins of many retroviruses. Our laboratory showed that the myristate + basic domain mediates plasma membrane targeting of HIV-1 Gag, thereby allowing Gag to function in the formation and budding of virions.
We then examined signaling proteins modified by the 16-carbon fatty acid palmitate. We showed that several Src-related proteins are dually fatty acylated with both myristate and palmitate. The palmitylation reaction is dynamic and reversible, and regulates the ability of Src family members to bind to membranes and participate in signaling. We have defined the molecular mechanisms whereby dual fatty acylation mediates intracellular protein trafficking, targeting to plasma membrane rafts, and intracellular signal transduction.
Our laboratory’s current efforts focus on exploring the mechanism of palmitoylation of secreted morphogens such as Hedgehog proteins and Wnt proteins. We have established assays for monitoring palmitoylation of Sonic hedgehog (Shh), have purified Hedgehog acyltransferase (Hhat), the enzyme that catalyzes attachment of palmitate to Shh, and have characterized the enzymatic mechanism and membrane topology of Hhat. In addition, we have exploited high throughput screening to identify Hhat inhibitors. These inhibitors are being tested for their ability to block the growth of cancers that are dependent on Shh signaling.