The ubiquitination-mediated proteolysis pathway is implicated in the control of the cell cycle. The ubiquitination process involves a three-step cascade that is driven by the enzymes El, E2, and E3. Two classes of E3 have been found to play significant roles in recognizing specific substrates important for cell cycle progression. One is the anaphase-promoting complex (APC) and the other is the Skpl -Cullin-F box protein (SCF) complex. Skp 1 is a highly conserved protein that associates with cullin-1, Rbxl and an F-box-containing protein to form the SCF complex. Apart from forming the SCF complex, Skpl was also found to function in kinetochore formation and activation. These imply that Skpl may have multiple functions in the cell. In my thesis research, the human Skpl protein and its int...[ Read more ]

The ubiquitination-mediated proteolysis pathway is implicated in the control of the cell cycle. The ubiquitination process involves a three-step cascade that is driven by the enzymes El, E2, and E3. Two classes of E3 have been found to play significant roles in recognizing specific substrates important for cell cycle progression. One is the anaphase-promoting complex (APC) and the other is the Skpl -Cullin-F box protein (SCF) complex. Skp 1 is a highly conserved protein that associates with cullin-1, Rbxl and an F-box-containing protein to form the SCF complex. Apart from forming the SCF complex, Skpl was also found to function in kinetochore formation and activation. These imply that Skpl may have multiple functions in the cell. In my thesis research, the human Skpl protein and its interactions with other components of the SCF complex were characterized. Skpl expressed in bacteria was found to be a non-spherical protein, denatured at 25°C, a fairly low temperature, and renatured when the temperature was lowered. Various methods, including cell free extracts, the yeast two-hybrid system, and expression in mammalian cells, were used to investigate the interaction between Skpl and its partners. I found that the N-terminal region of Skpl was important for binding to Skp2, cullin-1, and cyclin F. Similarly, the N-terminal region of cullin-1 was required for binding to Skp 1. Furthermore, novel Skpl-interacting proteins were identified using the yeast two-hybrid system to screen a cDNA library. These results provide an initial characterization of a protein that is essential for the degradation of many key cell cycle regulators.