Genes VII

The protein tyrosine kinases constitute a major class of oncoproteins, and fall into two general groups: transmembrane receptors for growth factors; and cytoplasmic enzymes. We have more understanding about the biological functions of the receptors, because we know the general nature of the signal transduction cascades that they initiate, and we can see how their inappropriate activation may be oncogenic. The normal roles in the cell of the cytoplasmic tyrosine kinases are not so well defined, but in several cases it appears that they provide catalytic functions for receptors that themselves lack kinase activity; that is, the activation of the receptor leads to activation of the cytoplasmic tyrosine kinase. We have a great deal of information about their enzymatic activities and the molecular effects of oncogenic mutations, although it has been more difficult to identify their physiological targets.

Receptors for many growth factors have kinase activity. They tend to be large integral membrane proteins, with domains assembled in modular fashion from a variety of sources. We discussed the general nature of transmembrane receptors and the means by which they are activated to initiate signal transduction cascades in 26 Signal transduction. The EGF receptor is the paradigm for tyrosine kinase receptors. The extracellular N-terminal region binds the ligand that activates the receptor. The intracellular C-terminal region includes a domain that has tyrosine kinase activity. Most of the receptors that are coded by cellular proto-oncogenes have a similar form of organization.

Dimerization of the extracellular domain of a receptor activates the tyrosine kinase activity of the intracellular domain. Various forms of this reaction were summarized previously in Figure 26.14. When the cytoplasmic domains of the monomers are brought into contact, they trigger an autophosphorylation reaction, in which each monomer phosphorylates the other.

A (generalized) relationship between a growth factor receptor and an oncogenic variant is illustrated in Figure 28.15. The wild-type receptor is regulated by ligand binding. In the absence of ligand, the monomers do not interact. Growth factor binding triggers an interaction, allowing the receptor to form dimers. This in turn activates the receptor, and triggers signal transduction. By contrast, the oncogenic variant spontaneously forms dimers that are constitutively active. Different types of events may be responsible for the constitutive dimerization and activation in different growth factor receptors.

The oncogene v-erb is a truncated version of c-erbB, the gene coding for the EGF receptor. The oncoprotein retains the tyrosine kinase and transmembrane domains, but lacks the N-terminal part of the protein that binds EGF, and does not have the C-terminus. The deletions at both ends may be needed for oncogenicity. The change in the extracellular N-terminal domain allows the protein to dimerize spontaneously; and the C-terminal deletion removes a cytosolic domain that inhibits transforming activity. There is also an activating mutation in the catalytic domain. So the basis for oncogenicity is the combination of mutations that activate the receptor constitutively.

The general principle that constitutive or altered activity may be responsible for oncogenicity applies to the group of growth factor receptors summarized previously in Figure 28.14. Another example of an activation event is provided by erbB2, which codes for a receptor closely related to the EGF receptor. An oncogenic form has a key mutation in its transmembrane region; this increases the propensity of the receptor monomers to form dimers.

Some proto-oncogenes code for receptors or factors involved in the development of particular cell types. Mutation of such a receptor (or growth factor) may promote unrestricted growth of cells of the appropriate type. The proto-oncogene c-fms codes for the CSF-I receptor, which mediates the action of colony stimulating factor I, a macrophage growth factor that stimulates the growth and maturation of myeloid precursor cells. c-fms can be rendered oncogenic by a mutation in the extracellular domain; this increases dimerization and makes the protein constitutively active in the absence of CSF-I. Oncogenicity is enhanced by C-terminal mutations, which could be inactivating an inhibitory intracellular domain.

The cellular action and basis for oncogenicity of the cytoplasmic group of protein tyrosine kinases is more obscure. The cytoplasmic group is characterized by the viral oncogenes src, yes, fgr, fps/fes, abl, ros. (c-Src is actually associated with membranes.) A major stretch of the sequences of all these genes is related, corresponding to residues 80-516 of c-src. This includes the SH2 and SH3 domains and the catalytic domain responsible for kinase activity. Presumably the regions outside this domain control the activities of the individual members of the family. In few cases, however, do we know the cellular function of a c-onc member of this group.

The paradigm for a cytoplasmic tyrosine kinase in search of a role is presented by the Src proteins. Since its isolation by Rous in 1911, RSV has been perpetuated under a variety of conditions, and there are now several "strains," carrying variants of v-src. The common feature in the sequence of v-src is that the C-terminal sequence of c-src has been replaced. The various strains contain different point mutations within the src sequence.

Proteins in the Src family were the first oncoproteins of the kinase type to be characterized. Src was also the first example of a kinase whose target is a tyrosine residue in protein. The level of phosphotyrosine is increased about 10 in cells that have been transformed by RSV. In addition to acting on other proteins, Src is able to phosphorylate itself.

Src proteins have several interesting features. Figure 28.16 summarizes their activities in terms of protein domains.

Both v-Src and c-Src are modified at the N-terminus. The N-terminal amino acid is cleaved, and myristic acid (a rare fatty acid of 14 carbon residues) is covalently added to the N-terminal glycine. Myristoylation enables Src proteins to attach to the cytosolic face of membranes in the cytoplasm. Most of the protein is associated with the cytoplasmic face of the endosomes, and it is enriched in regions of cell to cell contact and adhesion plaques.

Myristoylation is essential for oncogenic activity of v-Src, since N-terminal mutants that cannot be myristoylated have reduced tumorigenicity. The simplest explanation for the dependence of transformation on the membrane location of v-Src is that important substrates for Src are located in the membrane.

The major difference between v-Src and c-Src lies in their kinase activities. The activity of v-Src is ~20 greater than that of c-Src. The transforming activity of src mutants is correlated with the level of kinase activity, and we believe that oncogenicity results from phosphorylation of target protein(s). We do not know whether the increased activity is itself responsible for oncogenicity or whether there is also a change in the specificity with which target proteins are recognized.

Kinase activity plays two roles in Src function. First, attempts to identify a function for the phosphorylation in cell transformation have concentrated on identifying cellular substrates that may be targets for v-Src (especially those that may not be recognized by c-Src). A variety of substrates has been identified, but none has yet been equated with the cause of transformation. Second, the state of phosphorylation of Src itself may be important for its transforming activity.

Two sites in Src control its kinase activity. The c-Src protein is phosphorylated in vivo by the kinase Csk at tyrosine residue 527, which is part of the C-terminal sequence of 19 amino acids that is missing from v-Src. The v-Src protein is phosphorylated in vivo at Tyr-416, which is located in the catalytic domain. This position is not normally phosphorylated in vivo in c-Src.

The importance of these phosphorylations can be tested by mutating the tyrosine residues at 416 and 527 to prevent addition of phosphate groups. The mutations have opposite effects, as summarized in Figure 28.17:

• Mutation of Tyr-527 to the related amino acid phenylalanine activates the transforming potential of c-Src. The protein c-Src^Phe-527 becomes phosphorylated on Tyr-416, has its kinase activity increased ~10 , and it transforms target cells, although not as effectively as v-Src. Phosphorylation of Tyr-527 therefore represses the oncogenicity of c-Src. Removal of this residue when the C-terminal region was lost in generating v-Src contributes significantly to the oncogenic activity of the transforming protein.
• Mutation of Tyr-416 in c-Src eliminates its residual ability to transform. This mutation also greatly reduces the activity of the c-Src^Phe-527 mutant. It also reduces the transforming potential of v-Src, but less effectively. Phosphorylation at Tyr-416 therefore activates the oncogenicity of Src proteins.

Point mutations at other positions in c-Src support a correlation in which oncogenicity is associated with decreased phosphorylation at Tyr-527 and increased phosphorylation at Tyr-416. The state of these tyrosines may therefore be a general indicator of the oncogenic potential of c-Src. The reduced phosphorylation at Tyr-527 may be responsible for the increased phosphorylation at Tyr-416, which may be the crucial event. However, v-Src is less dependent on the state of Tyr-416, and mutants at this position retain transforming activity; presumably v-src has accumulated other mutations that increase transforming potential.

What is the function of c-Src; and how is it related to the oncogenicity of v-Src? The c-Src and v-Src proteins are very similar: they share N-terminal modification, cellular location, and protein tyrosine kinase activity. c-Src is expressed at high levels in several types of terminally differentiated cells, which suggests that it is not involved in regulating cell proliferation. But we have so far been unable to determine the normal function of c-Src.

The modulatory region of c-Src contains two motifs that are found in a variety of other cytoplasmic proteins that are involved in signal transduction: these may connect a protein to the components that are upstream and downstream of it in a signaling pathway. The names of these two domains, SH2 and SH3, reflect their original identification as regions of Src homology. We discussed their functions in 26 Signal transduction.

How is c-Src usually activated? Most mutations in the SH2 region reduce transforming activity (suggesting that the SH2 function is required to activate c-Src), and most mutations in SH3 increase transforming activity (suggesting that SH3 has a negative regulatory role). Figure 28.18 shows a more detailed autoregulatory model for the function of the SH2 domain. The state of phosphorylation at Tyr-527 is critical. In the inactive state, Tyr-527 is phosphorylated, and this enables the C-terminal region of c-Src itself to bind to the N-terminal SH2 domain. When an appropriate receptor tyrosine kinase (such as PDGF receptor) is activated, the autophosphorylation reaction creates a phosphopeptide sequence that binds to the c-Src SH2 region, releasing the region containing Tyr-527, which may be dephosphorylated. The following events are not entirely clear; one possibility is that this leads to the phosphorylation of Tyr-416, and activation of kinase activity. In this way, c-Src kinase activity responds to the activation of the receptor kinase. In any case, the oncogenic v-Src protein, of course, lacks Tyr-527 and is constitutively active.

Alternative ways for activating c-Src may be involved in some oncogenic reactions. For example, the polyoma middle T antigen activates c-Src by binding to the C-terminal region including Tyr-527 and prevents its phosphorylation. Some mutations in the SH2 domain of c-Src can activate the kinase activity (with oncogenic consequences), presumably because they prevent it from sequestering Tyr-527. Mutations in the SH2 and SH3 domains of c-Src can influence its specificity with regard to transforming different types of target cells, which suggests that these regions provide the connections to other (cell specific) proteins in the pathway.