Biochemistry; Biophysics; Molecular Biology; Morphogenesis; Schizophrenia; Stress, Physiological
WHRY Pilot Project Program Investigators
Research SummaryOur lab has a long-standing interest in understanding how growth factor and adhesion receptor signaling controls changes in cell shape and movement. We have pursued these studies primarily in three model systems: fibroblast migration, breast cancer cell invasion and metastasis, and the formation and stabilization of neuronal dendrites and dendritic spines.In each of these systems, we have developed biochemical approaches to elucidate signaling mechanisms, genetic approaches in mice to manipulate these signaling mechanisms, and a complementary collection of quantitative assays of cell migration, in vivo metastasis, electrophysiology, neuroanatomy, and animal behavior as functional readouts.
Extensive Research Description
Identification and characterization of Mediator-containing RNA polymerase II holoenzyme. As an undergraduate student with Henry Pitot at the University of Wisconsin, I helped characterize the gene encoding the barbiturate-induced aldehyde dehydrogenase from rat liver. This project exposed me to the compelling idea that an environmental cue, in this case phenobarbital, can dramatically change the gene-expression profile of the hepatocyte. My Ph.D. thesis research with Rick Young at the Whitehead Institute/MIT pursued my growing fascination with the complicated process of how genes get turned on. I identified an “initiation factor” required for RNA polymerase II to begin transcription of protein-coding genes in yeast. I demonstrated that this factor was part of a large complex, which we called the SRB complex (for suppressor of RNA polymerase B/II), contemporaneously discovered and named Mediator by Roger Kornberg’s lab. I went on to purify RNA polymerase II in complex with SRB complex/Mediator and a large subset of the general transcription factors. I demonstrated that this RNA polymerase II “holoenzyme” (a term I coined) appears to be the form most readily recruited to promoters in vivo and was responsive to transcriptional activators, a feature not observed with purified RNA pol II and general transcription factors alone. Together, my experiments revised models for how RNA polymerase II assembles onto gene sequences and how transcriptional activator proteins stimulate this process.
a. Dunn, T.J., Koleske, A.J., Lindahl, R., and Pitot, H.C. (1989) Phenobarbital-inducible aldehyde dehydrogenase in the rat. cDNA sequence and regulation of the mRNA by phenobarbital in responsive rats. J. Biol. Chem. 264:13057-13065.
b. Koleske, A.J., Buratowski, S., Nonet, M., and Young, R.A. (1992) A novel transcription factor reveals a functional link between the RNA polymerase II CTD and TFIID. Cell 69: 883-894.
c. Thompson, C.M., Koleske, A.J., Chao, D.M. and Young, R.A. (1993) A multisubunit complex associated with the RNA polymerase II CTD and TATA-binding protein in yeast.Cell 73: 1361-1375.
d. Koleske, A.J., and Young, R.A. (1994) An RNA polymerase II holoenzyme responsive to activators. Nature 368: 466-469. This letter was featured in News and Views: Carey, M. (2004) Simplifying the complex. Nature 368: 402-403.
Abl family kinases regulate cell shape and movement. The Abl (Abl1) nonreceptor tyrosine kinase was originally identified in the 1970s as a proto-oncogene associated with mouse and human leukemias, but the normal physiological function of the Abl family kinases was unknown. In work I began as a postdoc with David Baltimore at MIT that continues in my own lab, I used genetic and cell biological approaches to demonstrate that the Abl1/Abl and Abl2/Arg kinases play diverse roles as key regulators of cell morphogenesis and migration in tissues and cell types.In one early study we demonstrated that ablation of Abl1 and Abl2 in mice disrupts neuroepithelial cell morphogenesis, leading to defects in neurulation. Subsequent work from our lab demonstrated key requirements for these kinases in neuronal morphogenesis, neuronal dendrite stability, fibroblast migration, breast cancer cell invasion and metastasis, and macrophage engulfment of parasites. Our studies at the cellular level have identified key roles for Abl1 and Abl2 as mediators between cell surface adhesion and growth factor receptors and the coordination of adhesion dynamics and actin cytoskeletal rearrangements.
a. *Koleske, A.J., Gifford, A. M., Scott, M. L., Nee, M., Bronson, R. T., Miczek, K., and Baltimore, D. (1998) Essential roles for the Abl and Arg tyrosine kinases in neurulation. Neuron 21: 1259-1272. *Corresponding author
b. Miller, A.L., Wang, Y., Mooseker, M., and Koleske, A.J. (2004) The Abl-related gene (Arg) requires its F-actin:microtubule crosslinking activity to regulate lamellipodial dynamics during fibroblast adhesion. J. Cell Biol., 165: 407-419.
This was a featured article in JCB: Wells, W.A. (2004) Arg links actin and microtubules.J. Cell Biol. 165: 294-295
c. Lapetina, S., Mader, C.C., Machida, K., Mayer, B.J., and Koleske, A.J.(2009) Arg interacts with cortactin to promote adhesion-dependent cell edge protrusion. J. Cell Biol., 185: 503-19
This article was featured in the Nature Publishing Group’s Cell Migration Gateway.K. Bauman “Interactions at the front: Arg(uing) with cortactin”
d. Gil-Henn, H., Patsialou, A., Condeelis, J., and Koleske, A.J. (2013) Arg promotes invasion and attenuates proliferation of breast cancer in vivo. Oncogene, 32(21):2622-30.PMCID:PMC3473103
Regulation of Abl family kinase activity in normal cells and invasive breast cancer cells. One major focus of our work has been to understand how Abl and Arg translate signals from cell surface receptors into intracellular signaling pathways. In early work, we established a biochemical system using purified recombinant Abl family kinases to elucidate activation mechanisms.Our work showed that two conserved phosphorylation events mediate >10-fold activation of Abl and Arg kinase activities. Phosphorylation of a tyrosine in the SH2 domain kinase linker is mediated by autophosphorylation of the kinase in trans, while phosphorylation on the activation loop is mediated by Src family kinases. We also discovered that integrin adhesion receptors and growth factor receptors are potent activators of Abl family kinases in diverse cell types. We found that epidermal growth factor signaling through Src mediates Arg kinase activation in breast cancer cells, and this is a key trigger for invadopodium formation and invasive migration. We found that the Arg interacts with integrin beta 1 subunit tail via a series of discrete binding and phosphorylation events and we have mapped key interaction interfaces that are critical for these interactions. Our ongoing work is using structural approaches to understand the integrin:Abl2 activation mechanisms at atomic level resolution. We are also developing single molecule imaging approaches in cells to understand how growth factor and adhesion receptor engagement affects recruitment of Arg to the cell membrane and how these events relate spatiotemporally to changes in cytoskeletal structure and cell movement.
a. Tanis, K., Duewel, H.S., Veach, D., Bornmann, W. and Koleske, A.J. (2003) Two distinct phosphorylation pathways have additive effects on Abl family kinase activation. Mol. Cell. Biol. 23: 3884-3896.
b. Warren, M.S., Gourley, S.L., Lin, Y.-C., Simpson, M.A., Taylor, J.R., Greer, C.A. and Koleske, A.J. (2012) Integrin β1 signals through Arg to regulate postnatal dendritic arborization, synapse density, and behavior. J Neurosci. 32(8):2824-34. PMCID:PMC3313657
c. Mader, C.C., Oser, M., Magalhaes, M., *Condeelis, J., *Koleske, A.J., and Gil-Henn, H. (2011) EGF signaling through a Src-Arg-cortactin relay regulates breast cancer invasiveness by controlling invadopodia maturation and function. Cancer Research, 71(5):1730-41. PMCID:PMC3057139
*Corresponding authors – Hava Gil-Henn was an Associate Research Scientist in the Koleske lab at time of publication
d. Simpson, M.A., Bradley, W.D., Harburger, D., Parsons, M., Calderwood, D.A., and Koleske, A.J. (2015) Direct interactions with the integrin beta1 cytoplasmic tail modulate Abl2/Arg kinase activity. J Biol Chem, 290(13):8360-72. PMCID: PMC4375489
4. Regulation of dendrite, dendritic spine, and synapse development and stability. Building on our findings that Abl family kinases are key regulators of neuronal development and function, we have elucidated key biochemical pathways that control long-term dendrite and dendritic spine stability. In addition to our identification of upstream activators (see #3 above), this work has led us to identify the Rho inhibitor p190RhoGAP and actin polymerization and stability regulator cortactin as key interaction partners and substrates of Abl family kinases. Our work has revealed that many components of these pathways (integrins a3 and b1, Arg, p190RhoGAP, p120RasGAP, Rho, cortactin) are essential for long-term maintenance of normal synapse and dendrite structure. For example, although neurons develop normally in mice lacking integrin a3, b1, or Arg through postnatal day 21, they subsequently begin to lose synapses and dendrites, leading to significant synapse loss and dendritic regression by early adulthood. This neuronal destabilization is accompanied by significant deficits in behaviors that require proper hippocampal and prefrontal cortical function. Importantly, mutations or microdeletions affecting several of the proteins we have implicated in dendrite, spine, and synapse development and stability (integrins a3 and b1, Arg, p190RhoGAP, SHP-2, GluN2B) are associated with neurological and psychiatric disorders in humans underscoring the importance of our work for understanding human brain function. Because this work has highlighted the fundamental instructive role of extracellular matrix (ECM) in dendrite development, a major current focus is to identify key ECM molecules that regulate dendrite development and elucidating their mechanisms of action. We are also interested understanding how pathological states (e.g. chronic stress) impact these signaling mechanisms to trigger dendrite, dendritic spine, and synapse loss.
a. Moresco, E. M. Y., Donaldson, S., Williamson, A., and Koleske, A.J. (2005) Integrin-mediated dendrite branch maintenance requires Abelson (Abl) family kinases. J Neurosci., 25: 6105-6118
This was a featured article in J Neuroscience: (2005) EnAbling dendrite branching. J Neurosci 25: 26.
b. Gourley, S.L., Olevska, A., Warren, M.S., Taylor, J., and Koleske, A.J. (2012) Arg kinase regulates prefrontal dendritic spine refinement and cocaine-induced plasticity. J Neurosci. 32(7):2314-23. PMCID:PMC3386297.
c. Warren, M.S., Gourley, S.L., Lin, Y.-C., Simpson, M.A., Taylor, J.R., Greer, C.A. and Koleske, A.J. (2012) Integrin β1 signals through Arg to regulate postnatal dendritic arborization, synapse density, and behavior. J Neurosci. 32(8):2824-34. PMCID:PMC3313657
d. Kerrisk, M.E., Greer, C.A., and Koleske, A.J. (2013) Integrin α3 is required for late postnatal stability of dendritic spines, synapses, dendritic arbor complexity, and mouse behavior. J Neurosci. 33(16):6742-52. PMCID:PMC3711182
Mentoring, Education, and Science Policy. I am very strongly committed to career development of my trainees and others throughout the larger scientific community. With support of our work from NIH and private foundation grants, I have trained 15 Ph.D. students from diverse Yale Ph.D. programs (Genetics, Neuroscience, Molecular Biophysics and Biochemistry, M.D./Ph.D., Cell Biology, MCD-Biology), as well as 9 postdoctoral trainees, and all are still working scientists in an academia or industry.My trainees have gone on to Tenure Track/Staff Scientist positions in academia (Bar-Ilan University Medical School, Broad Institute/MIT, Emory, Hussman Institute/U. Maryland, U. Michigan, U. Nebraska, U. Texas-Southwestern, Old Dominion) or group leadership positions in Pharma/Biotech (Amgen, Caris Life Sciences, Constellation Pharmaceuticals). I am honored to serve as an active mentor for an exceptional group of trainees (6 predoctoral, 2 postdoctoral) currently in my lab. I also play an active role in science education. At Yale, I have participated in reworking how we teach introductory Biology, teaming with our partner departments (MCD-Biology, Ecology and Evolutionary Biology) to implement a unified first year biology curriculum. I also lead a graduate seminar in Molecular Biology and play various leadership roles in graduate education, currently serving as the Director of our combined BBS program. At the local level, I have hosted high school students to work in my lab and served as a judge for the Connecticut Science Olympiad. I have participated in the National Academies Summer Workshop on Science Education and adopted these best practices in the classroom. As a member of the ASCB Education Committee, I have organized symposia at the national meeting focusing on career development and promoting URM retention of undergraduate STEM majors. Finally, as member of the ASCB Public Policy Committee and Project 50, I have advocated for science and science funding. This has involved multiple advocacy meetings with U.S. Congress and Staff members on Capitol Hill, the annual “We Are Research” campaign during Nobel week, and encouraging letter writing to Congressional representatives.
- Warren, M.S., Gourley, S.L., Lin, Y.-C., Simpson, M.A., Taylor, J.R., Greer, C.A. and Koleske, A.J. (2012) Integrin ß1 signals through Arg to regulate postnatal dendritic arborization, synapse density, and behavior. J Neurosci. In press.
- Gourley, S.L., Olevska, A., Warren, M.S., Taylor, J., and Koleske, A.J. (2012) Arg kinase regulates prefrontal dendritic spine refinement and cocaine-induced plasticity. J Neurosci. In press.
- MacGrath, S.M. and Koleske, A.J. (2012) Cell Science at a glance: cortactin. J Cell Sci, In press.
- Mader, C.C., Oser, M., Magalhaes, M., *Condeelis, J., *Koleske, A.J., and Gil-Henn, H. (2011) EGF signaling through a Src-Arg-cortactin relay regulates breast cancer invasiveness by controlling invadopodia maturation and function. Cancer Research, 71(5):1730-41. *Corresponding Author
- Lin, Y.-C., and Koleske, A.J. (2010) Mechanisms that promote long-term synapse and dendrite stability: disruption in psychiatric and neurodegenerative disorders. Ann Rev Neurosci., 33:349-78.
- Gourley, S.L. Koleske, A.J., and Taylor, J.R. (2009) Loss of dendrite stabilization by the Abl-related gene (Arg) kinase regulates behavioral flexibility and sensitivity to cocaine. Proc. Natl. Acad. Sci. USA, 106(39):16859-64
- Bradley, W.D., and Koleske, A.J. Regulation of cell migration and morphogenesis by Abl family kinases: emerging mechanisms and physiological contexts. (2009) J. Cell Sci, in press.
- Lapetina, S., Mader, C.C., Machida, K., Mayer, B.J., and Koleske, A.J. (2009) Arg interacts with cortactin to promote adhesion-dependent cell edge protrusion. J. Cell Biol., 185: 503-19