Molecular Biology of Cancer Topics

Molecular BIology of Cancer Topics

Tyrosine Kinase Oncogenes

Neu Receptor Tyr Kinase

The Neu oncogene (aka erbB2, HER2) encodes a receptor tyrosne kinase similar to the epidermal growth factor receptor. In the rat, the oncogene has a missense mutation: Glu (GAG) substitutes Val (GTG). As a result of the Neu missense mutation, receptor aggregation occurs in absence of ligand. Oncogne Neu is thus constitutively activated and will increase the activity of proteins activated by Neu, for example phospholipase Cg., and induce the malignant transformation of cells.

Experimental Results:

DHFR/G8 cells (wild-type Neu) and B104 cells (Neu oncogene) were treated or not with a cross-linking agent that covalently binds proteins together. Neu protein was immunoprecipitated and resolved by SDS-PAGE. High molecular weight Neu dimers were detected in the wild type DHFR/G98 cells only when treated with the cross-linking agent. High molecular weight Neu dimers were detected in the oncogene B104 cells with and without crosslinking agent treatment. (Nature 330:230-231, 1989)

When untreated cells expressing none (NIH) wild type (NE19) and oncogenic Neu (B104) are immunoprecipitated with using an antibody for Neu, then resolved in a Western using phospho-tyrosine antibody, only the Neu from oncogenic cells is detected. If the same cells are immunoprecipitated with PLCg antibody and resolved in a Western using phospho-tyrosine antibody, both Neu and PLCg will be detected in oncogenic cells only. If the western is resolved using Neu antibody, Neu will be detected only in oncogenic cells, indicating Neu was associated with PLCg in this oncogenic cells. Data not shown here indicates all cell lines contain PLCg but only NE19 and B104 contain Neu. (EMBO J 10;2977-86, 1991)

Wild-type Neu is amplified in 28% of human breast cancer and 27% of human ovarian cancer patients, and the number of copies of the gene correlates inversly with survival time.

Herceptin (rhuMAb, HER2) is an antibody against Neu that inhibits Neu activity. According to Baselga et. al., recombinant humanized anti-HER2 antibody (Herceptin) enhances the antitumor activity of paclitaxel and doxorubicin against HER2/Neu overexpressing human breast cancer xenografts (Cancer Res. 58: 2825-2831, 1998).

In a Phase II study of weekly intravenous Herceptin in patients with HER2/Neu-overexpressing metastatic breast cancer, therapy caused tumor regression or stable disease in many of the treated patients.Without Herceptin, all patients probably would have had disease progression. (Baselga et al. J. Clin. Oncol. 14: 737-744, 1996)

Non-receptor Tyrosine Kinases

The Src family of non-receptor tyrosine kinases include Src, Yes, Fgr, Lck, Fyn, Lyn, and Hck. They associate with inner face of plasma membrane through a myristic acid lipid tail and contain SH3 and/or SH2 domains, a tyrosine kinase domain and tyrosines available for phosphorylation.

Src is regulated by phosphorylation of at least two tyrosines. Phosphorylation of Tyr 416 increases Src activity, while phosphorlation of Tyr527 decreases activity.

Src can be activated differently by the diverse signaling pathways in which it participates. Its SH2 domain binds phosphotyrosine of activated growth factor receptor/tyrosine kinase, then Src can be phosphorylated by the receptor tyrosine kinase. Alternatively, SH3 can bind other proteins associated with G-protein coupled receptors. Adaptor proteins such as GRB2 bind Src phosphotyrosines.

When Tyr 527 is phosphorylated, Src’s own SH2 domain binds to it, and the Src kinase domain is in the repressed, inactive conformation. When Tyr 527 is unphosphorylated, the Src tyrosine kinase domain is in the open, activated conformation and Tyr 416 can be phosphorylated to help activate Src.

Experimental results demonstrate the importance of Tyr 527 in Src regulation:

The SH2 domain of Src binds to phosphotyrosine 527 in vitro.
Mutation of Tyr 527 to Phe results in constitutive Src activation.
The 3’ end of v-src oncogene was found to be deleted, thus the Src protein product lacks Tyr 527, resulting in continuous activation of the kinase.

In addition to the C-terminal deletion (Tyr 527 is missing) that makes it oncogenic, the viral form of src (v-src) has several amino acid substitutions due to point mutations in the v-src oncogene.

Another oncogenic form of non-receptor tyrosine kinase is coded by the Philadelphia chromosome. Chromosomal translocation between bcr and c-abl genes create a fusion gene and thus a fusion protein. The translocation is t(9;22)(p34;q11). The c-abl proto-oncogene encodes a nonreceptor tyrosine kinase related to Src. Human patients with chronic myelogenous leukemia (CML), acute lymphocytic leukemia (ALL), and acute myelogenous leukemia (AML) exhibit the Philadelphia chromosome in the leukemic cells. Virtually all CML patients have the bcr-abl oncogene.

The bcr promoter sequences are required for bcr-abl -induced transformation to occur. In the fusion protein, the Bcr domain binds the SH2 domain of Abl, therefore Abl cannot bind its own phosphotyrosine (like Src Tyr 527) to shut itself off and remains constitutively active.

Experimental Results:

Radiolabeled lysates of Sf9 cells infected with wild-type baculovirus or P160 Bcr recombinant baculovirus were incubated with GST, GST-SH3, GST-SH2, GST-SH3/SH2 or anti-Bcr antibody. Afterwards bound proteins were resolved by SDS-PAGE. Bcr was detected bound to SH2 or anti-Bcr only in the Bcr recombinant lysates. (sorry I lost the reference)

Continue to "Ras" or take a quiz: [Q1].

Need more practice? Answer the review questions below.

Questions under construction

Continue scrolling to answers below.

Hey! DON'T PEEK!!! Finish the questions fist!

Answers under construction