The Inner Life of the Cell

http://www.youtube.com/watch?v=2-p-QajenM0&feature=related

Structural study of cell-cycle control proteins

: Structural basis of ubiquitylation

Current Opinion in Structural Biology 2002, 12:822–830

:Ubiquitin ligases: cell-cycle control and cancer

NATURE Reviews Cancer 2006, 6:369-381

The control of the cell cycle

Growth factor (mitogen)

Anti-proliferative signals

(CDK activating kinase)

Cell cycle control by ubiquitylation

(Structural study of SCF and APC)

Ubiquitin

The three dimensional structure of ubiquitin: contains 76 amino acids

Simplified view of the cell-cycle control system

Levels of cyclin expression during cell divisionare periodic1. This is the result of a constant syntheticrate coupled with a defined window in the cycle ofspecific proteolysis, which is executed by the ubiquitinproteasomesystem (UPS).

Cell cycle control of SCF ubiquitin ligase by proteolysis of Cdk inhibitor protein

P27 (CIP)

CKIs, negative-regulators of cyclin–CDK kinase complexes, are also targetedfor degradation by the UPS.

Three-layer regulation of the cell cycle

Therefore, the cell cycle is predominantly regulated by two types of post-

translational protein modification — phosphorylation and ubiquitylation.

Overview of the ubiquitin-proteasome pathway

B. The E3 mediates the transfer of ubiquitin from the E2 to the substrate protein by

promoting the formation of an isopeptide bond between the Ub carboxy-terminus and

specific lysine side chains on the substrate.

C. E3s bind both the protein target and a cognate E2 and have a central role in

conferring specificity to the ubiquitination pathway.

A. Ubiquitin-protein ligases (also known as E3s) act at the last step of a three-enzyme

cascade involving the ubiquitin-activating (E1) and ubiquitin-conjugating (E2) enzymes.

D. The mechanism by which they promote ubiquitination has not been well understood.

Ubiquitin ligase (E3) enzyme complex

HECT-type E3s catalyse ubiquitination

by first forming an E3–ubiquitin

thioester intermediate.

RING-type E3s do not appear to form

such an intermediate. They are

characterized by the presence of a RING

zinc finger domain that binds the E2.

Two distinct types of E3s

A. The SCF complexes are RING-type E3s

B. The largest family of ubiquitin–protein ligases.

The SCF (Skp1–Cullin–F-box protein) complexes

C. ubiquitinate a broad range of proteins involved in cell cycle

progression, signal transduction and transcription.

D. Deregulation of SCF-dependent proteolysis can contribute to

neoplastic transformation.

SCFSkp2 : Cdk-inhibitor p27Kip1

SCFFbw7 : cyclinE

SCFb-TrCP : b-catenin and IkB

Human SCF complexes with demonstrated E3 activity

The SCF complexes are RING-type E3s that consist of

A. Cul1 (776 residues),

B. Rbx1 (108 residues),

C. Skp1 (163 residues) and

D. F-box protein family (430 to.1,000 residues).

The composition of SCF complexes

Rbx1, which contains the RING domain, and Cul1 form a catalytic core

complex

that recruits a cognate E2F-box proteins are characterized by an amino-terminal 40-residue F-box motif

that binds Skp1 followed by protein–protein interaction modules such as

leucine rich repeats or WD-40 repeats that bind substrate.

invariable

variable

E3 components in the UPS are thought to be primarily responsible for the

specific recognition of a large number of target proteins. This requires both

specificity and versatility, which are provided by the existence of 500–1,000

different E3 ligases.

How is it possible to ubiqutinate various substrate?

B. In addition to multiple F-box proteins, most higher eukaryotes also contain

multiple homologues of the other SCF subunits, including two Rbx1 and five

cullin family members (paralogues) conserved from C. elegans to humans.

How is it possible to make various SCFs to ubiqutinate various substrate?

A. The large number of F-box proteins in eukaryotic genomes (at least 38 in

human) allows for the specific ubiquitination of a large number of

functionally and structurally diverse substrates

The schematic structures of SCF

Cell-cycle regulation by the SCF complex and APC/C

SCF

APC

Functions of the SKP1–CUL1–F-box-protein (SCF) complex

Cell-cycle regulation by the SCF complex and APC/C

The structure of Skp1 and Skp2 complex

Overall structure of the SCFskp2

The N-terminal domain of Cullin1

N-terminal tip of repeat 1 that is the Skp1-F boxSkp2 binding site

The C-terminal domain of Cullin1 and Rbx1

30 A° -wide groove

The Cul1 residues that contact Rbx1 are shown in light green, and the Rbx1 residues in pink.

Intermolecular b-sheet formed by Rbx1 and Cul1 C-terminal domain

The zinc-finger (RING) domain of Rbx1

Rigidity of Cul1 scaffold required for SCF function

The Cul1 linker mutant retains the ability to bind phosphorylated p27, in a manner dependent on the presence of Skp1, Skp2 and Cks1.

The SCFSkp2 complex with the wild-type (WT)

Cul1 (lane 1) but not the linker mutant Cul1

(lane 3) promotes the Cks1-dependent

polyubiquitination (Ubn ) of p27 in an in vitro

ubiquitination assay reconstituted with purified

components.

To startinvestigating the importance of the rigid architecture of the Cul1scaffold, we sought to construct a Cul1 mutant where the NTD andCTD interface is disrupted, and where the two domains are linkedby a flexible linker (Fig. 5a).

Model of the SCFSkp2–E2 complex

1 2 3

4 5 6

1 : prophase, 2 : pro-metaphase 3 : metaphase4, 5, 6 : early, mid, and late anaphase, respectively

Fixed HeLa cells were stained for DNA (blue), microtubules (green) and kinetochores (red)

The principal stages of mitosis in human cells and chromosome segregation

Regulation of mitosis by ubiqutin ligase APC (anaphase promoting complex)

(cullin-like)

(RING-finger domain)

Isolation of Native Human APC

APC was immunoprecipitated from

extracts of HeLa cells using CDC27

peptide antibodies.

Bound complexes were subsequently

eluted in

their native form with an excess of

antigenic peptide.The peptide was subsequently separated from

the eluted

protein by gel filtration chromatography

SDS–PAGE and silver staining analysis of the

resulting fractions revealed all known 11

subunits of human APCwhose identity was confirmed by

immunoblotting (not shown)

In the presence of purified

ubiquitin, E1 and E2 enzymes,

and ATP, the APC fractions were

able to ubiquitinate a

radiolabeled fragment of cyclin B

in a dose-dependent manner

Characterization of Native Human APC

Native electrophoreses of APC

Electron Microscopy of Negatively Stained APC

Diameter of 15 nm

140A° X 140A° x135A° in size

3D Model of the APC Obtained by Cryo-Electron Microscopy

Using this procedure, a 3D model of the APC with

a final resolution of 24 A° was generated.

Purified APC samples were imaged using

liquid nitrogen temperature electron

microscopy.

About 13,000 molecular images of

randomly orientated APC particles were

interactively collected from digitized

micrographs.

A first set of characteristic APC views was

obtained by multivariate statistical analysis

and automatic classification.

After angular reconstitution, a preliminary low

resolution

3D structure was derived.

Subsequently, the resolution of the structure was

reiteratively improved by generating large

number of reference images and performing

multiple cycles of multireference alignment,

automatic classification, and angular

reconstitution.