The Lilien-Balsamo Lab

Jack Lilien

Ph.D., University of Chicago, 1967; Postdoctoral Fellow, University of Pennsylvania, 1967-1969; Assistant, Associate and Full Professor, University of Wisconsin, 1969-1990; Professor and Chair, University of Wisconsin 1989; Professor and Chair, Clemson University, 1990-1994; Professor and Chair, Wayne State University, 1994-2000; Professor and Chair, University of Iowa, 2000-.

Janne Balsamo

Ph.D, University of Sao Paulo, Brazil, 1972; Post doctoral fellow, University of Wisconsin, Madison, 1973-1975; Project Associate, Senior Scientist, University of Wisconsin, Madison, 1976-1989; Research Professor, Clemson University, 1990-1994; Research Professor, Wayne State University, 1994-2000; Associate Research Scientist, University of Iowa, 2000-present.

Cell-cell and cell-substrate adhesion molecules play a crucial role in guiding cells and axons along their paths during tissue and organ formation and during formation of the intricate patterns of nerve connections. Our interest is focused on molecules in each of the three major families: integrins, cadherins and immunoglobulin superfamily (IgSF). Integrins function as αβ heterodimers to form heterophilic adhesions with extracellular matrix components such as fibronectin and laminin. Cadherins mediate homophilic, or cadherin-cadherin adhesions, as do members of the IgSF of cell-cell adhesion molecules. The cytoplasmic domains of these adhesion receptors interact with signaling molecules and adaptor molecules that propagate signals initiated by adhesive interactions, in some cases directly altering the function of the adhesion molecule and in other cases initiating signal cascades that alter cell function in many different ways. Our goals are to determine:

1. How adhesion molecules are regulated by extracellular cues: Development of the stereotypical arrangement of tissues and organs as well as axonal projections during embryogenesis requires the coordinated action of many different and overlapping molecular mechanisms, which ensure that cellular rearrangements follow the correct temporal and spatial paths. For example, growth cone guidance relies on many different adhesion receptors, including members of each of the three families mentioned above. The function of each of these adhesion molecules must be integrated into a coherent program for the growth cone to follow the appropriate trajectory. One extracellular guidance cue, the chondroitin sulfate proteoglycan Neurocan lines many axonal pathways in the developing central nervous system and regulates the function of both N-cadherin and β1-integrin (see Balsamo et al., 1995; Li et al., 2000). It is our hypothesis that axons are prevented from straying by this rapid and coordinate inactivation. Neurocan exerts its effect by causing release of an essential tyrosine kinase (Fer) from the cadherin cytoplasmic domain and its transfer to the cytoplasmic domain of β1-integrin (Li et al. 2000). The result of this is that β-catenin becomes hyperphosphorylated and the cadherin actin connection is broken and cadherin function is compromised.

Another essential secreted guidance cue, Slit, when bound to its receptor Robo repels growing axons. It is one of a combination of guidance cues that are essential for the proper formation of commisures at the midline in organisms as diverse as Drosophila and mice. We have recently discovered that one of the effects of Slit-Robo interaction is to silence or inactivate N-cadherin (Rhee et al. 2002). The mechanism through which this silencing occurs is through the formation of complex between Robo, N-Cadherin and non-receptor tyrosine kinase Abl resulting in the hyperphosphorylation of -catenin by Abl and loss of the cadherin cytoskeletal connection. It is interesting to note that the feature common to the two guidance cues Neurocan and Slit is the hyperphosphorylation of β-catenin which regulates cadherin function. This emphasizes the important role that β-catenin plays as a down stream target of many different guidance cues.

2. The molecular mechanisms through which adhesion molecule function is altered: At the onset and during migration cells and axons must react to environmental cues rapidly to maintain the appropriate trajectory. Such changes may be brought about through alterations in cadherin or integrin function, as opposed to expression. These epigenetic changes are mediated by effectors bound at the cytoplasmic domain of adhesion molecules. One cause of cadherin inactivation is severing its linkage to the cytoskeleton (Lilien et al. 2002). The linkage is mediated by β-catenin bound to the cytoplasmic domain of cadherin with α-catenin making the connection between β-catenin and actin. The phosphorylation of β-catenin on tyrosine residues regulates its interaction with cadherin and thus adhesion function. Two effectors regulate the phosphorylation of β-catenin, the non-receptor tyrosine kinase Fer and the tyrosine phosphatase PTP1B. Fer is bound indirectly to the cadherin cadherin cytoplasmic domain through the armadillo family member p120ctn (Xu et al. 2003), while PTP1B binds directly to the cytoplasmic domain of cadherin (Balsamo et al., 1996, 1998). In order to bind to cadherin PTP1B must be phosporylated at tyrosine 152 (Rhee et al., 2000). One role of Fer is to phosphorylate PTP1B (Xu et al. 2003), targeting it the cadherin cytoplasmic domain where it is regulates the dephosphorylation of β-catenin (Xu et al. 2003). , the non-receptor tyrosine phosphatase PTP1B, from its association with the cytoplasmic domain of cadherin. It is interesting to note that PTP1B also regulates the activity of integrin by activating the tyrosine kinase src, a requisite to integrin function (Arregui et al., 1998).

3. The molecular basis for demyelination in the peripheral nervous system: The smallest member of the IgSF, the myelin specific protein P0, is thought to mediate adhesion between successive membrane layers as the Schwann cell wraps around the axon. Mutations in P0 give rise to the progressive neurodegenerative disease, Charcot Marie Tooth disease. We have discovered that phosphorylation of essential residues in the cytoplasmic domain of P0 by the serine/threonine kinase PKC is essential for P0 function (Xu et al., 2000). Furthermore, the PKC target residues coincide with human mutations that result in demyelination. Thus, mutations which disrupt the targeting of PKC to the cytoplasmic domain, or mutations eliminating essential serine residues in the P0 cytoplasmic domain give rise to demyelinating disease. Continuing work on this problem involves characterizing the effectors or adaptors whose function and/or binding is affected by PKC-mediated phosphorylation and potential transcriptional targets for the signal cascade(s) initiated at the cytoplamsic domain of P0.

These problems have in common changes in signaling molecules associated with the cytoplasmic domains of the adhesion receptors. In each case we have used in vitro models in which the function of one or more adhesion molecules may be analyzed, coupled with strategies that perturb specific protein-protein interactions among the components associated with the cytoplasmic domain. One strategy is to introduce into cells mutant forms of molecules thought to regulate adhesion molecules, such as PTP1B. Another strategy has been to introduce into cells or tissues peptide competitors which prevent protein-protein interactions such as those essential for the binding of β-catenin to cadherin, or Fer to integrin. A third strategy has been to mutate residues in the cytoplasmic domains of the adhesion molecules thought to mediate the binding of essential regulatory or adaptor molecules. These combined strategies have been a powerful arsenal for addressing the function and significance of effector and adaptors associated with adhesion receptors.

Recent Publications:
2008 S.-H. Lee, Peng, I-F., Ng, S., Yanagisawa, M., Bamji, S.X., Elia, L., Balsamo, J., Lilien, J., Anastasiadis, P.Z., Ullian, E. and Reichardt, L.F. Synapses are regulated by the cytoplasmic tyrosine kinase Fer in a pathway mediated by p120catenin, Fer, SHP-2 and β-catenin. J.Cell Biol. In press.

2008 K. Bhalla, Beachem, M.A., Buchan, T., Guzauskas, G.F., Ladd, S., Bratcher, S.J., DuPont, B.R., Schroer, R.J., Balsamo, J., Lilien, J., and Srivastava, A.K. Alterations in CDH15 and KIRREL3 in patients with mild to severe cognitive impairment. Am. J. Human Gen. In press.

2007 J. Rhee, L. Buchan, T., Zukerberg, J. Lilien, and J. Balsamo. Cables Links Robo-Associated Abl Kinase to Cadherin-Associated β-catenin to Mediate Slit-Induced Modulation of Adhesion and Transcription. Nature Cell Biol. 9:883-892. Selected by "Faculty of 1000"

2007 A. Gaboreanu, R. Hrstka, J. Lilien, and J. Balsamo. Myelin Protein Zero/P0 Function Requires an Adaptor Protein Linking it to RACK 1 and PKCα. J. Cell Biol. 177:707-717

2006 G.S. Marrs, L. Fuller, R. Thangavel, T. Honda, M. Dailey, J. Balsamo, J. Lilien, and C. Arregui. Inactivation of β1-integrins Destabilizes Retinal Ganglion Cell Dendritic Arbors in situ. Mol. Cell. Neurosci. 32:230-241

2006 G. Davies Sala, M. V. Hernandez, J. Balsamo, J. Lilien, C. O. Arregui. Structural Determinants of Protein Tyrosine Phosphatase 1B (PTP1B) Targeting to Cell-Matrix Adhesion Sites. J. Cell Sci. 119:1233-1243. Selected by "Faculty of 1000"

2005 J. Lilien and J. Balsamo. Rapid Reversible Changes in Cadherin Function regulated by Tyrosine Phosphorylation of β-catenin. Curr. Opin. Cell Biol. 17:459-65

2004 G. Xu, Greer, P., Craig, A., Anastasiatis, P., Miller, M., Lilien, J., and Balsamo, J. Continuous Association of Cadherin with β-catenin Requires Phosphorylation of PTP1B by Fer. J. Cell Sci. 117:3207-3219.

2003 C. Arregui, T. Honda, B. Shah, M. Dailey, J. Balsamo, and J. Lilien. Inactivation of ß1-integrins Destabilizes Retinal Ganglion Cell Dendritic Arbors in situ. In preparation

2003 G. Xu, Greer, P., Craig, A., Anastasiatis, P., Miller, M., Lilien, J., and Balsamo, J. Continuous Association of Cadherin with ß-catenin Requires Phosphorylation of PTP1B by Fer. Submitted

2003 M. E. Shy, Jani, A., Krajewski, K., Lewis, R.A., Li, J., Shy, R.R., Balsamo, J., Lilien, J., Garbern, J.Y., and Kamholz, J. Phenotypic Clustering in MPZ Mutations. Brain 00324.R2

2002 G. Xu, C. Arregui, J. Lilien, and J. Balsamo. PTP1B Modulates the Association of ß-catenin with N-Cadherin through Binding to an Adjacent and Partially Overlapping Site. J. Biol. Chem. 277:49989-97.

2002 H. W. Sinn, J. Balsamo, J. Lilien and J.-C. Lin. Localization of the Novel Xin Protein to the Adherens Junction Complex in Cardiac and Skeletal Muscle During Development. Develop. Dyn. 225:1-13.

2002 J. Lilien, J. Balsamo, C. Arregui, G. Xu. Turn-off, Drop Out: Functional State Switching of Cadherins. Develop. Dyn. 224:18-29.

2001 W. Xu, M. Shy, J. Kamholz, L. Elferink, J. Lilien, and J. Balsamo. Mutations in the Cytoplasmic Domain of the Peripheral Myelin Protein P0 (MPZ) Abolish Adhesive Function and Reveal a Role for Protein Kinase C-Mediated Phosphorylation in Myelination. J. Cell Biol. 155:439-445.

2001 J-S. Rhee, J. Lilien, and J. Balsamo. The Binding of PTP1B to N-cadherin requires phosphorylation of tyrosine 152. J. Biol. Chem. 276:6640-6644.

2000 Pathre, P., T-C Leung, J. Lilien and J. Balsamo. PTP1B Regulates Neurite Outgrowth by PC12 Cells. Submitted.

2000 M. Shy, J. Balsamo, J. Lilien, and J. Kamholz. A Molecular Basis for Hereditary Motor and Sensory Neuropathy Disorders. Current Neurol. And Neurosci. Rep. 1:77-88.

2000 H. Li, T-C Leung, S. Hoffman, J. Balsamo and J. Lilien. Coordinate Regulation of Cadherin and Integrin Function by the Chondroitin Sulfate Proteoglycan Neurocan. J. Cell Biol. 149:1275-1288. 

2000 C. Arregui, P. Pathre, Lilien, J., and J. Balsamo. The Non-receptor Tyrosine Kinase Fer Mediates Crosstalk Between Cadherin and Integrin. J. Cell Biol. 149:1263-1273. 

1999 J. Lilien, C. Arregui, H. Li, and J. Balsamo. The juxtamembrane Domain of Cadherin Regulates Integrin-Mediated Adhesion and Neurite Outgrowth. J. Neurosci. Res. 58:727-734.

1998 C. Arregui, J. Lilien, and J. Balsamo. Impaired Integrin Mediated Adhesion and Signaling in Fibroblasts Expressing a Dominant-Negative Mutant PTP1B. J. Cell Biol. 143:861-873.

1998 J. Balsamo, C. Arregui, T-C. Leung, and J. Lilien. The Non-Receptor Protein Tyrosine Phosphatase PTP1B Binds to the Cytoplasmic Domain of N-cadherin and Regulates the Cadherin-Actin Linkage. J. Cell Biol. 143:523-532.

1997 J. Lilien, S. Hoffman, C. Eisenberg and J. Balsamo. ß-catenin is a Target for Extracellular Signals Controlling Cadherin Function: The Neurocan-GalNAcPTase Connection. Curr, Topics Dev. Biol. 15:161-189.

1996 J. Balsamo, T-C. Leung, H. Ernst, M.K.B. Zanin, S. Hoffman, and J. Lilien. Regulated Binding of a PTP1B-Like Phosphatase to N-Cadherin: Control of Cadherin-Mediated Adhesion by Dephosphorylation of ß-Catenin. J. Cell Biol. 134:801-813.

1995 J. Balsamo, H. Ernst, M.K.B. Zanin, S. Hoffman, and J. Lilien, The Interaction of the Retina Cell Surface N-Acetylgalactosaminylphosphotransferase with an Endogenous Proteoglycan Ligand Results in Inhibition of Cadherin Mediated Adhesion. J. Cell Biol. 129:1391-1403.