Scientist Boston Biomedical Research Institute
A.B. Smith College, Biochemistry,1984
Ph.D. University of California, Berkeley, Biochemistry 1989
Summary of Research
The actomyosin cytoskeleton plays a pivotal role in many diverse aspects of cell biology, yet morphologic changes can only occur if the activities of key cytoskeletal proteins are regulated spatially and temporally. We are interested in the biochemical events in the cell which coordinate the restructuring of the actomyosin cytoskeleton. Work in the lab is focused on the activation of myosin by regulartory light chain phosphorylation, using the model organism Dicyostelium discoideum. Dictyostelium is remarkably similar to many mammalian cells in its morphology and cell biology, and it offers a wide variety of experimental advantages. In these cells myosin is essential for cytokinesis and receptor capping, and plays an important role in chemotaxis. The Dictyostelium myosin light chain kinase MLCK-A is “unconventional”, as it is calcium/calmodulin independant. Instead, we have shown that it is regulated by phosphorylation in the activation loop by an upstream kinase. One project in the labratory is to characterize the upstream players in this signaling pathway. We have found that there are multiple MLCKs, in Dictyostelium. Thus a second project in the laboratory is the characterizing all of the signal transduction pathways involved regulatory light chain phosphorylation in nonmuscle cells. The presence of multiple MLCKs in nonmuscle cells is likely to be related tp the fact that myosin is involved in a diverse group of cellular processes, each of which is presumably regulated by distinct signaling pathways.
Gangopadhyay, S.S., Barber, A.L., Gallant, C., Grabarek, Z., Smith J.L. , and Morgan, K.G. (2003), Differential functional properties of CamKIIgamma Vairiants from Smooth Muscle. J. Biochem. .
Roelofs, J., Smith, J.L., and Van Haastert, P.J.M. (2003) Cyclic GMP Signaling: Different Ways to Create a Pathway. Trends Genet. Mar;19(3):132-4.
De la Roche, M.A., Smith, J.L., Roco M., CarrascoS., Merida I, Licate L., Cote, G.P, and Egelhoff, T.T. (2002), Dictyostelium Discoideum Has a Single Diacylglycerol Kinase Gene with Similarity to Mammalian Theta Isoforms. Biochem J., Dec 15;368(Pt3):809-15.
Bosgraff L., Russcher, H., Smith, J.L., Wessels, D., Soll, D.R., Van Haastert, P.J.M. (2002) A Novel cGMP Singlaing Pathway Mediating Myosin Phosphorylation and Chemotaxis in Dictyostelium. EMBO, 21:4560-70.
Jonathan M. Goldberg, Leonard Bosgraaf, Peter J.M. Van Haastert, and Janet L. Smith. (2002) Identification of four candidate cGMP targets in Dictyostelium, Proc. Natl. Acad. Sci., USA. 99:6749-54.
Singla, S.I., Hudmon, A., Goldberg, J.M., Smith, J.L., Schulman, H. (2001) Molecular Characterization of Calmodulin Trapping by Calcium/Calmodulin-dependent Protein Kinase II . J. Biol. Chem., 276, 29353-29360.
Silvera, L.A., J.L. Smith, and J.A. Spudich. (1998) MLCK-A, an unconcentional myosin light chain kinase from Dictyostelium, is activated by cGMP-dependant pathway. Proc. Natl. Acad. Sci. USA, 95, 13000-5
Smith, J.L., L.A. Silveria and J.A. Spudich.(1996) Activation of myosin light chain kinase A by phophorylation of threonine-166. EMBO J., 15, 6075-83.
Smith, J.L., L.A. Silveria and J.A. Spudich.(1996) Myosin light chain kinase A gene disruption in Dictyostelium. a role for MLCK-A in cytokinesis and evidence for multiple MLCKs. Proc. Natl. Acad. Sci. USA 93, 12321-6.
Smith, J.L., L.A. Silveria and J.A. Spudich.(1994) MLCK-A. Myosin light chain kinase A (dicyostelium Discoideum). In the Protein Kinase Factsbook. D.G. Hardie and S. Hanks, Eds. Academic Press, Inc. San Diego
Smith, J.L., A.B. Chapman and N. Agabian (1993) Trypanosoma vivax: evidence for only one RNA polymerase ll largest subunit gene in a trypanosome which undergoes antigenic variation. Experimental Parasitology, 76, 242-6.
Smith, J.L., J.R. Levin, N. Agabian. (1989) Molecular characterization of the Trypanosoma brucei RNA polymerase l and lll largest subunit genes J. Biol. Chem., 264,18091-9.
Smith, J.L., J.R. Levin, C.J. Ingles, and N. Agabian. (1989) In trypanosomes the homolog of the largest subunit of RNA polymerase ll is encoded by two genes and has highly unusual C-terminal domain structure Cell, 56,815-27.
Maintenance and Cell Care of AX3
Making HL-5 (Dicty Media)
Dicty Flag-Tag Western Blot Protocol
Whole Cell PCR (from Lisa Kreppel)
Knock Out Candidates: Frozen Stocks, Genomic DNA, and PCR
Total DNA Preperation for Dicty
cAMP Pulse Radioactive Experiment
SPERA Assay: Plaque Expansion Assay
Con A Stimulation
Making Dicty Stocks
Synchronous Morphogensis on Filter
Making Western Samples Directly from Dicty Cultures
E. Coli Protein Induction
TOPO TA Cloning
Transformation of CaCl2 Cells
Making CaCl2 Competent Cell Stock
Making DH Competent Cell Stocks and Transformation
Rapid Ligation Kit
Cos 7 Cells Transfection
Cos 7 Cells Harvesting
Extending a Genomic Sequence With Sequencing Databases
Using Adobe Photoshop to Scan Figures
Solution, Gels, Etc.
Making PAGE Gels
Making 6X DNA GEL loading buffer
Making SDS Buffer
Making Urea Gels