Publication list via PubMed

Introduction

Research in our laboratory is currently focused on the regulation of gap junctions by tyrosine protein kinases and novel, interacting cellular proteins. Gap junctions are one of four major types of intercellular junctions that are involved in establishing intercellular adhesion and communication. Gap junctions are unique because they enable the direct interchange of small molecules (<1000 daltons) between the cytoplasms of adjacent cells. This activity provides a form of intercellular signaling that is essential for the proper functioning of a variety of normal tissues. Hexameric complexes of connexins (connexons or hemichannels) in adjacent cell plasma membranes create intact gap junctions by the docking of the connexons across the extracellular space. There are twenty one connexin genes in the human genome, which encode the connexin proteins. Connexins are embedded in the plasma membrane by four transmembrane domains, which create two extracellular regions and three intracellular, cytoplasmically-located domains (see Figure 2). The C-terminal, cytoplasmic region of connexins often contains sites and/or elements that mediate the regulation or signaling capabilities of gap junctions. Our work is currently conducted in new Cancer Research Center laboratories located in the Biosciences Building located on the School of Medicine campus in Kakaako. This research has been supported by grants from the National Cancer Institute, National Institutes of Health (5 RO1 CA052098-18) and the American Heart Association.

Regulation of Connexin43 by Oncoprotein Tyrosine Kinases

One of our research efforts investigates the molecular mechanisms by which oncogenes and their encoded proteins regulate cell growth and induce the development of malignant cells. One of the largest classes of oncogenes is represented by the tyrosine protein kinases, which contain either transmembrane receptors (e.g.-EGF, PDGF) or cytoplasmic, non-receptor enzymes (e.g.-Src, Fyn, Yes, Lck). Tyrosine kinases act directly by phosphorylating cellular substrate proteins and/or indirectly by activating signal transducing proteins downstream of the tyrosine kinases, such as Ras and MAP kinase. These signaling pathways ultimately lead to the stimulation of cell growth and the modification of cell behaviors, such as increased cell migration, invasion, and tumor angiogenesis.

We have focused on how tyrosine kinases disrupt the function of the most ubiquitously expressed gap junction protein, connexin43 (Cx43). Because gap junctions permit the direct intercellular exchange of ions, small metabolites, and possibly, growth regulatory molecules, it has been suggested that connexins may exert growth suppressive effects. Thus, the loss of growth control and the development of malignant tumors may result, in part, from the disruption of gap junctional communication caused by activated tyrosine kinase oncoproteins found in tumors. We examined the effects of two tyrosine kinases, viral Src (vSrc) and the EGF receptor, on Cx43 phosphorylation and gap junction function.

We discovered that the vSrc tyrosine kinase profoundly disrupted intercellular gap junctional communication in fibroblast cells co-expressing vSrc and Cx43. This biological effect was associated with the phosphorylation of the cytoplasmically-located COOH tail of Cx43 at two specific sites, tyrosine 247 (Y247) and tyrosine 265 (Y265). Our research suggested that Cx43, through a proline-rich motif in its C-terminal region, may associate initially and directly with the SH3 domain of vSrc. This is followed by the phosphorylation of Cx43 at Y265, which may support the interaction with the SH2 domain of vSrc. We have proposed that the possible hierarchical phosphorylation of the second site at Y247 may lead to the closure of the gap junctions (Figure 1). Additional electrophysiological studies have suggested that vSrc induces channel closure by reducing the probability of open channels rather than decreasing the macroscopic conductance of gap junction channels.

Figure 1. A model for the interaction of v-Src with Cx43.
In this model, the binding of Cx43 to v-Src is dependent initially upon a SH3 domain interaction followed by SH2 domain interactions, which are important for v-Src–induced phosphorylation of Cx43 at the Y265 site and the subsequent phosphorylation at the Y247 site, leading to closure of the Cx43 channel. PXXP denotes the P274–P284 proline-rich sequence of Cx43 that interacts with the SH3 domain of v-Src. For simplicity, gap junction channels are depicted as cylinders. PM denotes the plasma membrane.

Parallel work investigating the effects of the activated EGF receptor (EGFR) on Cx43 gap junctions yielded surprisingly different results. Although activation of the EGFR in epithelial cells was associated with a profound, but transient depression in gap junctional communication and the increased phosphorylation of Cx43, phosphorylation occurred only on serine sites, in marked contrast to vSrc. These results indicated that Cx43 was not phosphorylated directly by the activated EGFR. Instead, we discovered that the increased phosphorylation was caused by MAP kinase that is activated downstream of the EGFR in a Ras-dependent manner. We identified S255, S279, and S282 as targets of the activated MAP kinase.

The sites in Cx43 that are targeted by the Src and EGF receptor tyrosine kinases and protein kinase C are summarized in Figure 3.

Figure 2. Phosphorylation sites identified in Cx43.
The phosphorylation sites in Cx43 that are targeted by the Src tyrosine kinase, the activated EGR receptor tyrosine kinase, and protein kinase C are indicated. The brackets indicate the proline-rich region in Cx43 that mediates binding to the SH3 domain of vSrc.

Kanemitsu, M.Y. and Lau, A.F. (1993) EGF stimulates the disruption of gap-junctional communication and connexin43 phosphorylation independent of TPA-sensitive PKC: the possible involvement of MAP kinase. Molecular Biology of the Cell, 4:837-848.

Warn-Cramer, B.J., Lampe, P.D., Kurata, W.E., Kanemitsu, M.Y., Loo, L.W.M., and Lau, A. F. (1996) Characterization of the MAP kinase phosphorylation sites on the connexin43 gap junction protein. Journal of Biological Chemistry, 271: 3779-3786.

Lampe, P.D., TenBroek, E., Burt, J.M., Kurata, W.E, Johnson, R.G., and Lau, A.F. (2000) Phosphorylation of connexin43 on serine368 by protein kinase C regulates gap junctional communication. Journal of Cell Biology, 149:1503-1512.

Lin, R., Warn-Cramer, B.J., Kurata, W.E., and Lau, A.F. (2001) v-Src phosphorylation of connexin43 on tyr247 and tyr265 disrupts gap junctional communication. Journal of Cell Biology, 154:815-827.

Cottrell, G.T., Lin, R., Warn-Cramer, B., Lau, A.F., and Burt, J.M. (2003) Mechanism of v-Src and mitogen-activated protein kinase-induced reduction of gap junction communication. American J. of Physiology: Cell Physiology, 284: C511-C520.

Lampe, P. and Lau, A. F. (2004) The effects of connexin phosphorylation on gap junctional communication. The International Journal of Biochemistry and Cell Biology, 36:1171-1186.

Warn-Cramer, B. J. and Lau, A. F. (2004) Regulation of gap junctions by tyrosine protein kinases. BBA Biomembranes-Special Issue: The Connexins (editor Jean Claude Herve), 1662:81-95.

Lau, A.F. (2005) c-Src: Bridging the gap between phosphorylation- and acidification-induced gap junction channel closure. Science STKE 2005, 291:pe33.

Lin, R., Martyn, K. D., Guyette, C. V., Lau, A. F., and Warn-Cramer, B. J. (2006) v-Src tyrosine phosphorylation of connexin43: regulation of gap junctional communication and effects on cell transformation. Cell Communication and Adhesion, 13:199-216.

Moreno, A. and Lau, A. F. (2007) Gap junction channel gating modulated through protein phosphorylation. Progress in Biophysics and Molecular Biology, 94:107-119.

Regulation of Connexin43 by CIP75, a Novel UBA-UbL Domain-Containing Protein

The degradation of connexin43 (Cx43) has been reported to involve both lysosomal and proteasomal degradation pathways; however, very little is known about the mechanisms regulating these Cx43 degradation pathways. Using yeast two-hybrid, GST pull-down, and co-immunoprecipitation approaches, we identified a novel Cx43-interacting protein of about 75 kDa, CIP75. Laser confocal microscopy showed that CIP75 is located primarily at the endoplasmic reticulum (ER), as indicated by the calnexin marker, with Cx43 co-localization in the perinuclear region (Figure 3). CIP75 belongs to the UbL (Ubiquitin-like)-UBA (Ubiquitin-associated) domain-containing protein family with an N-terminal UbL domain and a C-terminal UBA domain. The UBA domain at the C-terminus of CIP75 is the main element mediating the interaction with Cx43, whereas the CIP75-interacting region in Cx43 resides in the PY motif and multiphosphorylation sites located between Lys²⁶⁴ to Asn³⁰². Interestingly, the UbL domain, located at the N-terminus of CIP75, interacted with the S2/RPN1 and S5a/RPN10 proteins of the 19S regulatory subunit of the 26S proteasome complex. Overexpression experiments suggested that CIP75 is involved in the turnover of Cx43 as measured by a significant stimulation of Cx43 degradation and reduction in its half-life with the opposite effects on Cx43 degradation observed in CIIP75 siRNA knock-down experiments. These data suggested a working model where CIP75 may function as an adaptor or shuttle factor to facilitate the dislocation of misfolded or otherwise damaged Cx43 from the ER and transfer to the proteasome for degradation in the process known as ER-associated degradation or ERAD (see Figure 4).

Figure 3. CIP75 and Cx43 co-localize at the ER.
NRKe cells were transiently transfected with Flag-CIP75. The subcellular localization of CIP75 (green, A and F) and Cx43 (red, B and G) with the ER marker calnexin (blue, D and I) was visualized by LSCM. CIP75 expression causes increased levels of cytoplasmic Cx43 to varying degrees (top-lower levels, bottom-higher levels) where CIP75 co-localizes with Cx43 (yellow, C and H). CIP75 and Cx43 co-localization occurs at or around the ER (E and J). Scale bar: 10µm.

Figure 4. A working model for the interaction of CIP75 with Cx43 in the ER leading to the proteasomal degradation of Cx43.
Cx43, possibly ubiquitinated and located in the membrane of the ER, may interact with the UBA domain of CIP75, which may be involved in the dislocation of Cx43 from the ER. The UbL domain of CIP75 mediates the interaction with the Rpn1 and/or RPN10 proteins located in the 19S regulatory subunit of the proteasome. These interactions may facilitate the degradation of misfolded or damaged Cx43 by the 26S proteasome.

Li, X., Su, V., Kurata, W.E, Jin, C., and Lau, A.F. (2008) A novel Cx43-interacting protein, CIP75, which belongs to the UbL-UBA protein family, regulates the turnover of connexin43. Journal of Biological Chemistry, 283:5748-5759.

Regulation of Connexin43 by the RabGAP, CIP85

The yeast two-hybrid screen was also utilized to identify a novel Cx43-interacting protein of 85-kDa, CIP85, which contains a single TBC, SH3, and RUN domain, in addition to a short coiled coil region (Figure 5).  Homologues containing this unique combination of domains were found in human, D. melanogaster, and C. elegans.  CIP85 mRNA is expressed ubiquitously in mouse and human tissues with the highest expression in mouse brain and testis.  In vitro interaction assays and in vivo co-immunoprecipitation experiments confirmed the interaction of overexpressed or endogenously-expressed CIP85 with Cx43.  In vitro interaction experiments using CIP85 mutants with in-frame deletions of the TBC, SH3, and RUN domains indicated that the SH3 domain of CIP85 is primarily involved in its interaction with Cx43.  Conversely, analysis of Cx43 mutants with proline to alanine substitutions in the two proline-rich regions of Cx43 revealed that the
P₂₅₃LSP₂₅₆ motif (see Figure 2) is an important determinant of the ability of Cx43 to interact with CIP85.  Laser-scanning confocal microscopy showed that CIP85 co-localized with Cx43 at the cell periphery, particularly in areas reminiscent of gap junction plaques.  The functional importance of the interaction between CIP85 and Cx43 was suggested by the observation that CIP85 appeared to induce the turnover of Cx43 through the lysosomal pathway.

Figure 5. A model for the interaction of CIP85 with Cx43 at the plasma membrane.
CIP85 contains three major domains: TBC, SH3, and RUN. CIP85 interacts with Cx43 through its SH3 domain. The TBC domain of CIP85 may function as a GAP for small GTPase Rab proteins. The function of the RUN domain is not clearly defined.

Lan, Z., Kurata, W. E., Martyn, K. D., Jin, C., and Lau, A. F.  (2005)  A novel Rab GAP-like protein, CIP85, interacts with connexin43 and induces its degradation.  Biochemistry, 44:2385-2396.

Inhibitors of Oncoprotein Tyrosine Kinases found in Hawaii's Natural Products

The elucidation of signal transduction pathways that regulate cell proliferation and tumor angiogenesis offers numerous potential molecular targets for intervention by novel chemicals and small molecules.  In collaboration with the Natural Products and Cancer Biology Program chemists (Drs. Heimscheidt, Tius, and Williams), we have initiated an effort to identify novel compounds from natural product extracts or organically-synthesized that may inhibit the activity of selected oncoprotein kinases, which are involved in either the regulation of cell proliferation or tumor angiogenesis.  The Src tyrosine kinase (activated in a wide spectrum of human tumors), MAP kinase (stimulates cell growth), the vascular endothelial growth factor (VEGF) receptor tyrosine kinase (involved in tumor angiogenesis), and the cyclin D/Cdk4 kinase (regulates G1-S phase transition of the cell cycle) have been targeted for development.  In vitro assays for the Src and MAP kinases were developed successfully in the laboratory and are currently deployed in a central, shared screening laboratory to detect natural product inhibitors of these oncoprotein kinases.  Efforts are underway to complete the development an assay based upon the VEGF tyrosine kinase receptor.  These combined studies may identify novel lead compounds that may be developed as useful therapeutic or chemopreventive agents for cancer and other diseases.

Current Laboratory Members

Vivian Su, Ph.D.
Postdoctoral Researcher

Kimberly Cochrane, M.S.
Graduate Student, Cell and Molecular Biology Graduate Program

Wendy Kurata, M.S
Research Associate V

Former Laboratory Members

Postdoctoral Researchers: Gary Goldberg, Ph.D., Lakshmi Sivaraman, Ph.D., Margaret Dice, Ph.D., Bonnie Warn-Cramer, Ph.D., Lisa Pierce, Ph.D., Alison Glazier, Ph.D., Wade T. Kiyono, M.D., Teresa Samson, Ph.D., Zheng Lan, Ph.D., Xinli Li, Ph.D.

Graduate Students: Thomas Rayson, M.S., David Crow, Ph.D., Farial Sabrina, Frank Mukaida, M.S., Robril Tingcang, M.S., Martha Kanemitsu, Ph.D., Kendra Martyn, Ph.D., Lenora Loo, Ph.D., Andrea Demain, Chengshi Jin, Ph.D., Linda Flores, Rui Lin, Ph.D., Alison Walls, M.S.

Rotating Graduate Students: Le Ma, Martha Kanemitsu,Lenora Loo, Kendra Martyn, Robbie Tingcang, Zhao Liang Lu, Rui Lin, Ben Chang, Leanna Bartram, Linda Flores, Kenneth Takeuchi, Edith Margarian, Alison Walls, Jaime Horton

Undergraduate Students (MARC/MBRS/Howard Hughes Medical Institute):
Felix Ungacta, Andrea Demain, Edward Chargualaf, Shyla Penaroza, Creighton Tuzon, Dacia Brooks, Traci Kuwaye, Skye Chang, Nel Venzon, Kristine Ito-Smith, Meredith Cardenas

Other Undergraduate and Graduate Research Rotation Students: Darwin Chang, Elena Gerbaudo, Gayle Suzuki, Lori Kurashima, Maria-Louisa Andres, Stefan Moisyadi, Carl Sasaki, Chris Bjornson, James Palmer, Wayne Kimoto, JoAnne Furuya, Chaoquan Yin, Gregory Suh, Meredith Hermosura, Chengshi Jin, Janice Lai, Rhiana Lau, Carrie Guyette, Kelly Lock, David Veal, Lana Young, Dorhyun Johng, Andrew Knutson, Katharine Lau

High School CURE Awardee Summer Students: Jill Harunaga, Kiana Frank, Andrew Knutson, Lisa Harunaga, Carolynn Kitamura