Role of Tumor-Related Mutations in Zip Kinase in Controlling Actin Cytoskeleton
Poster Number
36
Format
Poster Presentation
Faculty Mentor Name
Doug Weiser
Faculty Mentor Department
Biological Sciences
Abstract/Artist Statement
Reversible phosphorylation of the type II myosin light chain 2 (MLC2) is a critical regulatory mechanism for controlling type II myosin and the actin cytoskeleton. Zipper Interacting Protein Kinase is a regulator of Myosin Light Chain 2 through phosphorylation of the MLC2, which is critical for proper myosin and actin cytoskeleton contraction and other important cellular processes including morphogenetic cell movements during development, smooth muscle contraction, and tumor cell invasion. ZIPK and ROCK regulate myosin phosphatase, which is responsible for dephosphorylating the MLC2. This is achieved by ZIPK/ROCK phosphorylating Mypt1 (myosin phosphatase target subunit 1) at the conserved threonines 696 and 850 which ultimately inhibits myosin phosphatase activity. As a result, when ZipK is inactivated, the cell begins to show irregularity such as disorganization of the cell membrane as well as improper functioning of actin and myosin. This would eventually lead to an incomplete mitosis cycle and cause apoptosis. In tumors from human patients, three mutations have been found in ZipK: T112M, D161N and P261S. We created these constructs by site directed mutagenesis and then transfected Hela cells to view under a confocal microscope. The D161N mutant Hela cell exhibited less actin and myosin organization compared to our wild type ZipK cells that show strong stress fiber organization. The D161N phenotype may be a result of a loss of function or dominant negative mutation. To verify which mutation is responsible we will perform a Phosphorylation Assay to verify the phosphorylation level of our mutant compared to our wild type. The wild type should exhibit high phosphorylation level. If D161N is a dominant negative, it should express low phosphorylation level. Testing on Zebrafish can further investigate whether it is a dominant negative or a loss of function. In the future, we will carry out similar experiments with the T112M and P261S mutants.
Location
DeRosa University Center, Ballroom
Start Date
25-4-2015 10:00 AM
End Date
25-4-2015 12:00 PM
Role of Tumor-Related Mutations in Zip Kinase in Controlling Actin Cytoskeleton
DeRosa University Center, Ballroom
Reversible phosphorylation of the type II myosin light chain 2 (MLC2) is a critical regulatory mechanism for controlling type II myosin and the actin cytoskeleton. Zipper Interacting Protein Kinase is a regulator of Myosin Light Chain 2 through phosphorylation of the MLC2, which is critical for proper myosin and actin cytoskeleton contraction and other important cellular processes including morphogenetic cell movements during development, smooth muscle contraction, and tumor cell invasion. ZIPK and ROCK regulate myosin phosphatase, which is responsible for dephosphorylating the MLC2. This is achieved by ZIPK/ROCK phosphorylating Mypt1 (myosin phosphatase target subunit 1) at the conserved threonines 696 and 850 which ultimately inhibits myosin phosphatase activity. As a result, when ZipK is inactivated, the cell begins to show irregularity such as disorganization of the cell membrane as well as improper functioning of actin and myosin. This would eventually lead to an incomplete mitosis cycle and cause apoptosis. In tumors from human patients, three mutations have been found in ZipK: T112M, D161N and P261S. We created these constructs by site directed mutagenesis and then transfected Hela cells to view under a confocal microscope. The D161N mutant Hela cell exhibited less actin and myosin organization compared to our wild type ZipK cells that show strong stress fiber organization. The D161N phenotype may be a result of a loss of function or dominant negative mutation. To verify which mutation is responsible we will perform a Phosphorylation Assay to verify the phosphorylation level of our mutant compared to our wild type. The wild type should exhibit high phosphorylation level. If D161N is a dominant negative, it should express low phosphorylation level. Testing on Zebrafish can further investigate whether it is a dominant negative or a loss of function. In the future, we will carry out similar experiments with the T112M and P261S mutants.