CDC4 and Endometrial Cancer The Role of Ubiquitylation in Tumorigenesis Luke Dong

Ubiquitin ligases (E1, E2, E3) SCFCDC4 (an E3 ligase) Overview Ubiquitin’s role Ubiquitin ligases (E1, E2, E3) SCFCDC4 (an E3 ligase) CDC4 and Cyclin E CDC4 mutations and Cancer

Ubiquitin Ubiquitin plays the vital role of tagging proteins for destruction by proteasomes when they need to be destroyed. The timely destruction of specific proteins propagates the cell cycle by allowing the next set of proteins to do their thing and end processes that have performed their purpose. E1 proteins capture free ubiquitin and brings them to E2. E2 proteins tag proteins with the ubiquitin for destruction. E3 proteins provide scaffolding for E2 to target specified substrates. We will focus on E3

Ubiquitin pathways

SCFCDC4 Construct The SCF (Skip ,Cullin, F-box) construct brings the substrate (e.g. Cyclin E or c-Myc) to the Ubc (E2 protein) to be ubiquitylated so a proteosome can destroy it. CDC4 contains two fundamental domains: the F-box, which mediates binding to the SCF core, and the substrate-binding domains, which can consist of one of several protein–protein interaction motifs. Ubc provides activated ubiquitin for ubiquitylation of Cyclin E Figure 4: Ratchets and Clocks: The Cell Cycle, Ubiquitylation, and Protein Turnover (© Nature Publishing Group 2003)

SCFCDC4 Construct CDC4 The SCF (Skip ,Cullin, F-box) construct brings the substrate (e.g. Cyclin E or c-Myc) to the Ubc (E2 protein) to be ubiquitylated so a proteosome can destroy it. CDC4 contains two fundamental domains: the F-box, which mediates binding to the SCF core, and the substrate-binding domains, which can consist of one of several protein–protein interaction motifs. Ubc provides activated ubiquitin for ubiquitylation of Cyclin E Figure 4: Ratchets and Clocks: The Cell Cycle, Ubiquitylation, and Protein Turnover (© Nature Publishing Group 2003)

Note that as Fbxw7/hCdc4 levels increase, Cyclin E levels drop off Note that as Fbxw7/hCdc4 levels increase, Cyclin E levels drop off. E2 (T392A) and E2 (S396A) are mutant E2 proteins which, as you’ll notice, are not affected by increasing levels of Fbxw7/hCdc4. Klotz, K. et al., SCFFbxw7/cCdc4 targets cyclin E2 for ubiquitin-dependent proteolysis. Exp. Cell Res. (2009).

In vitro, Cyclin E2 is ubiquitinized only in presence of the SCFFbxw7/hCdc4 complex. Klotz, K. et al., SCFFbxw7/cCdc4 targets cyclin E2 for ubiquitin-dependent proteolysis. Exp. Cell Res. (2009).

From CDC4 to Cancer Ying Meei Tan et al., The Fbxw7/hCdc4 tumor suppressor in human cancer. Cancer Letters 271 (2008) 1-12.

CDKs and Cyclins Recall that CDKs drive the cell cycle along by phosphorylating transcription factors, post-transcription factors, and other signaling proteins. CDKs are activated only when bound to an appropriate cyclin.

Cyclin E and CDK2 Cyclin E comes in to play immediately after the restriction point, preparing the cell to enter S phase. Some roles include: promoting centrosome duplication initializing histone synthesis regulation of replication S phase transcription Over expression of Cyclin E leads to accelerated G1. Cyclin D’s role is differentiation in G1 while Cyclin E promotes proliferation in S phase. But when G1 is shortened or eliminated, the cell fails to differentiate effectively. So, more proliferation but less functionality = problem. Note also that CDK2 is first activated by Cyclin E and later by Cyclin A. If Cyclin E is occupying all the CDK2, then Cyclin A struggles to play out its role in early S phase. - this leads to further replication problems. Example: Cyclin A promotes Orc1 localization to centrosomes where Orc1 prevents Cyclin E-dependent reduplication of both centrioles and centrosomes in a single cell division cycle. Figure 8.8 The Biology of Cancer (© Garland Science 2007)

SCFCDC4 in Drosophila Drosophila Eye d: wild-type f: CDC4 mutant Arrows: secondary pigment cells Arrowheads: tertiary pigment cells Moberg, K. et al., Archipelago regulates Cyclin E levels in Drosophila and is mutated in human cancer cell lines. Nature, 413 (2001), 311-316.

Mutations of CDC4 Black arrows are missense mutations; Red arrows are nonsense mutations. Nonsense mutations result in a STOP codon and are pretty straight forward as to how they produce cancerous phenotypes, but what’s really interesting is the location of the missense mutations. - 18 out of 19 missense mutations studied in this paper occurred in the WD40 repeats region of the protein. - many of the WD40 mutations eliminate specific arginines that may be involved in substrate-binding specificity. What are WD repeats? - The WD40-repeat domain of Cdc4 recognize phosphorylated serine and threonine containing peptides. - Cdc4 contains 8 repeated WD40 domains which is unique (most WD40 proteins contain 7) - http://pawsonlab.mshri.on.ca/index.php?option=com_content&task=view&Itemid=64&id=190 Dros Rajagopalan, H. et al. Inactivation of hCDC4 can cause chromosomal instability. Nature 428, 77-81 (2004).

CDC4 structure Blue: F-Box Green: WD40 repeats Orange (+ some blue): substrate (e.g. cyclin E)

hCDC4-/- human cells phenotype d: Blue = centromeres of chromosome 18; Red = centromeres of chromosome 7 e & f: Green = centrosomes; Red = spindles Note that in d, many centromeres are present when only two copies should exist. Note that in e and f, several spindle poles are present when only two poles should exist. Both phenotypes will cause aneuploidy. Rajagopalan, H. et al. Inactivatino of hCDC4 can cause chromosomal instability. Nature 428, 77-81 (2004).

Cyclin E and hCDC4 Fbxw7/hCDC4 mutations occur in a diverse set of human cancers including blood, breast, bile duct, colon, endometrium, stomach, lung, ovary, pancreas, and prostate. Overall mutation frequency is only ~6%. In endometrial cancers, however, the frequency is over 16%. The Fbxw7/hCDC4 function is also, however found to be suppressed in blood, breast, colon, esophagus, skin ovary, brain, and lung cancers among others. In most cases, Fbxw7/hCDC4 gene alterations were accompanied by a coordinate LOH of the remaining wt allele. In one study, 100% of endometrial cancer patients with Fbxw7/hCDC4 mut exhibited lymph node metastatis (compared to only 25% in wt Fbxw7/hCDC4 patients). This suggests Fbxw7/hCDC4’s role in aggressive cancer development. In culture, Fbxw7/hCDC4 inactivation results in micronuclei and chromosomal instability, a hallmark of cancer. Note that hCDC4 mutations do not always affect Cyclin E levels and Cyclin E levels do not always correlate with hCDC4 mutation, so what I’ve talked about is just one of many possible mechanisms of tumorigenesis by hCDC4 mutation. Recall that hCDC4 has many known tumorigenic proteins and new targets are still being found.

From CDC4 to Cancer

Future Direction Characterization and mechanisms of mutations for specific substrates? Role in DNA damage response? Other substrates?

Questions?