The Src Kinases

For a review on the Src kinases, see:

G. S. Martin, "The Hunting of the Src", Nature Reviews Molecular Cell Biology, vol 2, pages 467-475 (2001).

 

In 1911 Peyton Rous isolated from chickens a virus that when injected into healthy birds rapidly produced tumors. Now known as the Rous sarcoma virus, this was the first cancer-causing retrovirus to be isolated. Like all retroviruses, the Rous sarcoma virus has a simple genetic structure, with just three genes (gag, pol and env) that code for proteins which are needed to replicate the viral genome and to encapsulate it for further transmission. A fourth gene, v-src (for viral sarcoma) codes for a protein that on its own can induce in cultured cells the morphological and behavioral changes that characterize tumor cells. The v-src gene is responsible for the tumor causing potential of the virus and was the first of many tumor-causing genes, or oncogenes, to be isolated from viruses. It is now known that many of these genes have normal counterparts in animal genomes, including humans. The protein encoded by the cellular counterpart of v-Src, the c-Src protein, participates in the signal transduction pathways of receptors that regulate cell growth in animal cell. The oncogenic properties of the v-Src protein arise from disruptions in an internal control mechanism that normally prevents the activation of the protein in the absence of external signals.

 

The Src protein was the first TYROSINE KINASE to be discovered. Tyrosine kinases play cricical roles in signaling between cells in multi-cellular animals. An important aspect of these signaling mechanisms is the recognition of PHOSPHOTYROSINE by small protein modules known as SH2 domains (Src homology domains) which were first discovered in the Src proteins. The first step in such mechanisms is the activation of cell surface receptors (such as the insulin receptor, or epidermal growth factor (EGF) receptor) by extracellular ligands:

 

The activation of the receptor results in the activation of tyrosine kinases, which generate phosphotyrosine residues inside the cell. These phosphotyrosine residues act as "beacons" which attract signaling proteins that contain SH2 domains to the receptors:

The Src kinase has a structure composed of two peptide binding domains, in addition to a catalytic kinase domain. One of the peptide binding domains is the SH2 domain, and the other is an SH3 domain, which recognizes polyproline helices. The N-terminal region is myristylated, so the protein is associated with the cell membrane. Our lab determined the first crystal of an SH2 domain (that of v-Src).

 

The form of Src kinase that is encoded by Rous Sarcoma virus (v-Src) is constitutively active. In contrast, normal cellular Src (c-Src), is usually inactive until appropriately activated. We would like to understand how the sequence differences between v-Src and c-Src (shown below) results in the activation of v-Src.

The Src kinases are normally kept off by an autoinhibitory interaction between a phosphotyrosine-binding module known as the SH2 (Src-homology 2) domain, located within these proteins before the catalytic kinase domain, and a C-terminal phosphotyrosine residue (Tyr 527). The first structures of the inactive forms of the Src kinases Hck (determined by my group) and c-Src (determined by Stephen Harrison, HHMI, Harvard University, and his coworkers) revealed an architecture that was surprising, because the internal engagement of the SH2 domain by phosphorylated Tyr 527 occurs on the distal surface of the catalytic domain of the kinase, about 40 from the catalytic center. Neither the SH2 domain nor the SH3 domain (another modular binding domain present in the Src kinases) blocks the active site directly. Nevertheless, when the SH2 and SH3 domains are internally bound up the catalytic activity of the Src kinases is markedly reduced, and just how this happens is what wed like to understand.

There are 9 Src kinases in the human genome. These proteins are all closely related to each other, and share the same regulatory mechanism. Our crystallographic work has been focused on the Src kinase Hck (Hematopoietic cell kinase). One of our crystal structures of Hck in the inactive form is shown above.

Based on our crystal structures, as well as those determined by others, we have developed a reasonably good idea about the conformational transitions that the Src kinases undergo as they turn on and off:

We are using molecular dynamics simulations and mutational analyses to probe further into this question. We have carried out nanosecond timescale molecular dynamics simulations of c-Src and Hck, as well as simulations in which we artificially drive conformational changes in localized parts of these proteins. These simulations suggest that an important feature of the regulatory mechanism is the ability of the connector between the SH2 and SH3 domains to function as an "inducible snap lock," a segment of structure that is flexible except when it is locked into place by specific interactions at both ends. Our simulations show that when the SH2 and SH3 domains are bound to their internal targets within the protein, the connector between them becomes rigid and couples these domains dynamically. This coupling, which is dependent on tail phosphorylation, impedes the ability of the kinase domain to undergo breathing motions that are required for the catalytic center to rearrange into an active conformation. Presumably, upon activation by dephosphorylation of the tail these domains are released and are then also free to interact with cellular targets. The picture below shows changes in dynamical correlations in the protein when residues in the SH2-SH3 linker are replaced by glycine. For more information, click here.

In order to test ideas generated by the simulations we make mutations in the Src protein and study the effects of these mutations on the proper regulation of the kinase. Activated forms of Src kinases are difficult to overexpress and purify, perhaps because they kill cells through uncontrolled phosphorylation of other proteins. Our mutational analyses therefore rely on an assay for Src kinase activity that is based on the ability of unregulated Src kinases to kill the fission yeast Schizosaccharomyces pombe (this work is in collaboration with Giulio Superti-Furga, European Molecular Biology Laboratory).

S. pombe cells  

 

The graph on the right shows growth curves for S. pombe expressing empty vector (blue; normal growth). Expression of c-Src alone arrests growth (magenta), because yeast does not contain the enzyme Csk, that phosphorylates the tail of c-Src and inactivates it. Co-expression of Csk and c-Src (pink curve) results in normal growth, because tail phosphorylation by Csk inhibits Src. This system allows us to screen a large number of mutants to study how they alter the regulatory properties of the kinase. We are also using this system to study the Abl tyrosine kinase.

 

 

 

Using this system we have shown that if the connector linking the SH2 and SH3 domains of c-Src is made more flexible (for example, by replacing residues within this linker by glycine) this results in the activation of c-Src and Hck. Our computer simulations show that glycine residues in the connector break the dynamic correlations between the SH2 and SH3 domains, which presumably loosens up the protein and allows activating transitions to occur. A long term goal of this project is to use fluorescence spectroscopy, hydrogen exchange and NMR to characterize the dynamics of the Src kinases experimentally.