Structure and function of human MAT1, the assembly/targeting factor of CDK-activating kinase

MAT1 functions as the assembly/targeting factor of the CDK-activating kinase (CAK). To identify the specific domains of MAT1 required for CAK assembly, protein binding studies were performed both in vitro and in cells. We demonstrated that both the RING finger motif and the cyclin-like box of MAT1 are dispensable for the interaction of MAT1 with CDK7-cyclin H complexes. Both the coiled-coil region and the hydrophobic domain are required. These results indicate two non-contiguous domains are required for CAK assembly. This data, provided additional information regarding the precise domains of MAT1 involved in CAK formation, and provided a rationale for the selection of specific MAT1 mutants for subsequent functional studies. To determine the therapeutic potential of MAT1 blockade in cancer cells, a series of MAT1 constructs were engineered into the retroviral vector, and tested in terms of cell growth parameters. These studies demonstrated that the MAT1 constructs exerted little, if any, inhibitory effects on cell growth, DNA synthesis, protein synthesis, cell cycle kinetics, or site-specific CAK kinase activity in both human pancreatic cancer MIAPaCa2 and human osteosarcoma MG63 cells. In contrast, the blockade of cyclins with G1 cyclin constructs displayed marked reduction in cell growth. It is suggested that MAT1 may be of some utility in the inhibition of normal cells in certain proliferative diseases, it does not appear to represent a strategic locus for exerting control of cancer cells.; In a search for MAT1-interacting proteins using the two-hybrid system, MCM7, a newly characterized member of a family of DNA licensing factors, was identified. The physical interaction between MAT1 and MCM7 was confirmed in yeast cells and with in vitro protein binding assays. Further studies showed that the RING finger domain of MAT1 is not required for the observed interaction with MCM7, while the C-terminal domains of MAT1 is shown to be indispensable. Immunoprecipitation of MCM7 in human osteosarcoma MG63 cells demonstrated that MCM7 associates with the CAK complexes. These results suggest the multifunctional CAK complexes may coordinate DNA replication through physical interaction with MCM7, a DNA licensing factor.