Cynthia Brame's Research Interests
Cynthia Brame is Associate Professor of Biology at Centenary College.
Protein kinases are a ubiquitous family of enzymes that catalyze transferral of the terminal phosphate from ATP to a specific amino acid on a protein substrate. The eukaryotic protein kinase superfamily consists of eight subfamilies, one of which is the CK1 family of protein kinases. CK1s are ubiquitous and abundant protein kinases that regulate many physiological events, including cell proliferation and differentiation, chromosome segregation, and circadian rhythm. In humans, disruption of normal CK1 function has been linked to neurodegenerative diseases and cancer (1).
I am interested in structure/function relationships within the CK1 kinase domain. CK1s share features with other members of the kinase superfamily: a small N-terminal lobe comprised primarily of beta-helices (the beta lobe; shown in magenta), a larger lobe composed primarily of alpha-helices (the alpha lobe; shown in blue), and a Mg2+/ATP binding pocket at the juncture of the two lobes (2, 4). They also, however, exhibit several CK1-specific features, including a long activation loop that extends across the alpha lobe. In other kinase families, phosphorylation within the activation loop has been shown to activate kinase activity through shifts in protein structure that stabilize the active site (3). CK1s are constitutively active, meaning that they do not require phosphorylation for activity. Some evidence suggests, however, that they do undergo phosphorylation within the kinase domain and that this phosphorylation inhibits kinase activity (4,5).
In collaboration with Dr. Lucy Robinson at the LSU Health Sciences Center in Shreveport, I am currently investigating the hypothesis that specific phosphorylation events within the kinase domain inhibit CK1 activity. We use YCK2, a CK1 from budding yeast, as a model CK1, and use several approaches to investigate our hypothesis, including direct work with student researchers.
Working with Students
Students in BIOL 313 (Genetics) pursue a project in which they use bioinformatics tools and molecular biology techniques to generate, clone, and test the effect of mutant YCK2 alleles. This work has resulted in identification of several potential regulatory phosphorylation sites within the YCK2 kinase domain as described in the posters by McMahen et al. and Pahls et al. below. Students who pursue the project further in summer research use a combination of genetics, biochemistry, and microscopy to extend their findings. Jordan Day (Biology 2010) and Jessica Miller (Biology 2010) both presented the results of their summer work at the 2009 meeting of the American Society of Biochemistry and Molecular Biology; their posters are also shown below. In collaboration with Troy Messina, we are also modeling the effect of phosphorylation on CK1 structure in silico.
Investigation of a conserved tyrosine residue in a yeast casein kinase 1 protein kinase
Investigation of the Conserved RD Pocket in the CK1 protein kinase family
Does Phosphorylation of a Conserved Tyrosine Regulate CK1 Activity?
Does Phosphorylation of an Activation Loop Serine Inhibit CK1 Activity?
Investigation of a conserved serine residue in a in a yeast casein kinase 1 protein kinase
Knippschild U, Gocht A, Wolff S, Huber N, Lohler J, and Stoter M. The casein kinase 1 family: participation in multiple cellular processes in eukaryotes. Cellular Signaling 17 (2005) 675-689.
Hanks SK and Hunter T. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification. The FASEB Journal 9 (1995) 576-596.
Nolen B, Taylor S, and Ghosh G. Regulation of protein kinases: Controlling activity through activation segment conformation. Molecular Cell 15 (2004) 661-675.
Carmel G, Leichus B, Cheng X, Patterson S, Mirza U, Chait B, and Kuret J. Expression, purification, crystallization, and preliminary X-ray analysis of casein kinase 1 from Schizosaccharomyces pombe. The Journal of Biological Chemistry 269 (1994) 7304-7309.
Cegielska A, Gietzen K, Rivers A, and Virshup D. Autoinhibition of casein kinase 1 epsilon (CKIe) is relieved by protein phosphatases and limited proteolysis. The Journal of Biological Chemistry 273 (1998) 1357-1364.