Src family kinases (SFKs) are nonreceptor tyrosine kinases that act as key mediators of cellular signal transduction (12
). The nine SFK members, Src, Yes, Fyn, Hck, Lck, Lyn, Blk, Fgr, and Yrk, play crucial roles in cellular proliferation, survival, migration, and growth factor and cytokine stimulation pathways (26
). All SFKs share a similar domain arrangement, consisting of SH3, SH2, and kinase (SH1) domains as well as a unique domain and a membrane-targeting SH4 region at the N terminus (11
). Crystal structures have shown that the catalytic activity of SFKs is tightly regulated by autoinhibition. The SH3 domain binds to a polyproline region in the linker between the SH2 and kinase domains, and the SH2 domain binds to a phosphotyrosine residue (Tyr527 in avian c-Src) near the C terminus. Kinase activation can be achieved by displacing one or all of the autoinhibitory interactions (52
All SFKs are myristoylated at the N terminus (47
). Myristoylation occurs cotranslationally and is catalyzed by the enzyme N
-myristoyl transferase (NMT) (19
). The 14-carbon saturated fatty acid myristate is covalently attached to the N-terminal glycine residue via an amide bond, making myristoylation an essentially irreversible modification (44
). Myristoylation is necessary but not sufficient to anchor a protein to the membrane, and membrane binding of myristoylated proteins requires a second signal. For Src, the second signal is a polybasic cluster of amino acids that interacts with acidic phospholipids on the inner leaflet of the membrane bilayer (34
). Nearly all other SFKs are instead modified by attachment of the 16-carbon saturated fatty acid palmitate to cysteine residues 3 and 5 or 6 at the N terminus (48
). Myristoylation and palmitoylation together form a “dual signal” motif that targets SFKs to membranes.
Membrane binding is crucial for cellular functions mediated by Src and other SFKs. Nonmyristoylated forms of Src are cytoplasmic and cannot induce cellular transformation (14
). Membrane localization of c-Src has been shown to be important for dephosphorylation of Tyr527 and for mitotic activation of c-Src kinase activity (8
), presumably because the phosphatase that acts on Tyr527 is membrane bound. Myristoylation has also been proposed to play a role in regulating nuclear transport of c-Src (17
For some myristoylated proteins, the myristate moiety can exist in two different conformational states, either sequestered inside a hydrophobic pocket within the protein or exposed and available for membrane binding (44
). Binding to a ligand or another protein can cause a switch from one state to another, resulting in membrane association or dissociation. “Myristoyl switch” mechanisms have been identified in a variety of myristoylated proteins, including recoverin and HIV-1 Gag (4
). In the c-Abl tyrosine kinase, a “myristoyl phosphotyrosine” switch is operative. Myristate binds within a hydrophobic pocket at the base of the c-Abl kinase domain, docking the SH2 domain against the kinase domain in such a way that it prevents activation of the kinase by phosphotyrosine ligands (22
). A similar pocket is predicted to exist at the base of the c-Src kinase domain, raising the possibility that c-Src in the autoinhibited form might be capable of binding its own N-terminal myristate group in a manner similar to that of c-Abl (16
). To date, only nonmyristoylated, N-terminally truncated forms of c-Src have been crystallized, and the position of the myristate within the full-length c-Src protein is not known. A recent study provided support for the existence of a potential myristate binding pocket within c-Src (16
). Exogenous addition of myristate to the Tyr527-phosphorylated form of nonmyristoylated c-Src induced chemical shift changes in the nuclear magnetic resonance (NMR) spectra for both the protein and the fatty acid. However, the site of myristate binding was not determined. Thus, it is still not known how or if myristate regulates c-Src kinase activity and whether the predicted myristate binding pocket functions within c-Src in a manner similar to that of c-Abl.
In this study, we directly analyzed the role of myristoylation in regulating c-Src kinase activity and tested whether residues in the predicted myristate binding pocket contribute to myristate binding and/or c-Src activity. Here we show that myristoylation plays a positive role in regulating c-Src kinase activity. In contrast to c-Abl, nonmyristoylated c-Src exhibits reduced kinase activity both in vitro and in cells. We also made the surprising finding that the myristoylation status of c-Src determines its intracellular stability by regulating c-Src ubiquitination and degradation of the E3 ligase Cbl. Lastly, we analyzed the role of the predicted myristate binding pocket at the base of the c-Src kinase domain. Mutations in the pocket region resulted in decreased kinase activity and, with the exception of one mutation (Thr456Ala), had no effect on membrane binding of c-Src. We concluded that c-Src kinase activity is regulated by myristoylation, but in a different manner from that of c-Abl, and that a “myristoyl switch” is unlikely to be operative within c-Src.