“Cyclin I is involved in the regulation of cell cycle progression”

- Saleh Sarwar

Introduction:

Cyclin

Cyclin + Cdk = Regulation of cell cycle

By fluctuating levels

Cyclin I Member of cyclin family

Studied Cyclin box

PEST-motif

Sequence similarity to G1 and G2 cyclins

Partners with cdk5

Family of proteins = Cyclin box

Background:

Cyclin I role Correlated = Proliferative activity + angiogenesis

Increased expression = Terminal growth arrest

Unclear role in cell cycle progression

Some = no role as RNA level doesn’t fluctuate.

Hypothesis:

If cyclin I has role in cell cycle progression

Protein level should oscillate

Expression inhibition = no progression

Aims:

1- To investigate whether protein level of cyclin I fluctuates throughout cell cycle

2- To see whether cyclin I gets ubiquinated then degraded via proteosome and role of PEST motif in it.

3- To detect invivo association of cyclin I and cdk 5

4- To investigate role of cyclin I in cell cycle progression

Methods and Results:

1st Aim: Investigating protein level fluctuation throughout cell cycle:

HeLa cells + double thymidine block + collected diff stages/phases categorization = SDS PAGE+ Imunobloting

2nd Aim: Proteolysis, Ubiquination and PEST motif role:

Part 1 : Proteosome degradation:

pDsRed-Cyclin I

Lactacystin

HEK293T cells# of DsRed-Cyclin I positive cells increased

DsRed-Cyclin I protein level increased

Part 2: Ubiquitination

HEK293T cells

Over expressed with 1- Flag-Tagged Cyclin I

2- HA-tagged-Ubiquitin

Co expressionAnti-HA antibody detected at High MW

Part 3: PEST motif role in Proteolysis

A single change in last experiment

PEST motif deletion mutant

Anti-HA antibody still at high MW

3rd Aim: Investigating Invivo association of Cyclin I and cdk5

HEK293T cells 1- Flag-tagged cyclin I2- HA-tagged cdk5

Copercipitation detected

Immunopercipatation

4th Aim : Investigating role of cyclin I in cell cycle progression

HeLa Cells

siRNA for Cyclin I

Monitoring by WST-1 Assay

Cell proliferation prevented

Cell cycle profile determination by flow cytometry

Role in progression around G2/M

- The siRNA transfection of HeLa-Fucci cells confirmed results of flow cytometry.

Discussion:

1- Cyclin I no role in cell cycle progression = was proven wrong

2- PEST has no role in degradation so should be mediated through some other regions

3- Cyclin I + cdk5 = found throughout cells but whether it does cell cycle regulation by activating cdk 5 or by some other ways.