Our Science – Kuehn Website

Michael R. Kuehn, Ph.D.

Laboratory of Protein Dynamics and Signaling Senior Investigator Laboratory of Protein Dynamics and Signaling National Cancer Institute at Frederick 1050 Boyles Street, Bldg. 560, Rm. 12-44 Frederick, MD 21702-1201 Phone:   301-846-7451 Fax:   301-846-1666 E-Mail:   mkuehn@mail.nih.gov

Biography

Dr. Kuehn did his Ph.D. research with Norman Arnheim at Stony Brook and conducted postdoctoral research with Rudolf Jaenisch at the Heinrich-Pette-Institut in Hamburg, Germany, and with Nobel laureate Martin Evans at the University of Cambridge, England. He was an assistant professor at the University of Illinois College of Medicine before joining the NCI's Experimental Immunology Branch in 1991. In 2002, Dr. Kuehn joined the Laboratory of Protein Dynamics and Signaling at NCI-Frederick.

Research

Genetic Control of Vertebrate Embryonic Development

My research program is focused on basic mechanisms underlying the development of the mammalian embryo, with a goal toward understanding how aberrant regulation of developmentally important signaling pathways leads to human disease. Using retroviral insertional mutagenesis, we identified two developmentally essential genes, Nodal and Senp1, and are now characterizing these in detail. Our ongoing work on Nodal, a transforming growth factor (TGF)-β like secreted signaling molecule, has helped establish its role as a critical regulator of early vertebrate development. Recent work on Senp1, a protease regulating the dynamic process of post-translational modification by the ubiquitin-like protein SUMO, has revealed a role in placental development. Another approach we have taken to identify developmentally important genes is based on the hypothesis that critical regulatory molecules are regulated by ubiquitin mediated protein degradation, and can be identified by their interaction with E3 ubiquitin ligases, the component of the ubiquitination pathway conferring specificity. N4BP1 and N4BP3, two novel, developmentally expressed proteins identified in a yeast two hybrid screen as interaction partners for the E3 ubiquitin ligase Nedd4, are now being characterized further because of the potential involvement of each in tumorigenesis.

Our recent work on Nodal signaling in mouse embryonic development has focused on the analysis of a targeted mutant allele with reduced function (a hypomorph). Embryos heterozygous for this hypomorphic allele and a null allele develop past the block at gastrulation seen in null homozygotes, but then display a variety of developmental abnormalities. Analysis of these defects has allowed us to establish a critical role for Nodal signaling in patterning the anterior/posterior (AP) and left/right (LR) body axes, and in development of the midline mesendoderm and gut endoderm. However, the generalized reduction of activity in each of the many domains of Nodal expression provides no insight into where and when Nodal is required for these developmental events. To dissect the various temporal and spatial domains of Nodal function further, we have generated a conditional mutant (floxed) allele, which we are using in conjunction with transgenic strains expressing Cre recombinase in various regions of the developing embryo.

We have identified Senp1 as a second gene mutated by retroviral insertion. Loss of Senp1 function results in increased steady state levels of the sumoylated forms of a number of proteins, as well as decreased levels of free SUMO, and leads to placental defects and embryonic death just past mid-gestation. Current work is focused on identifying and characterizing sumoylated proteins that show increased levels in mutants. We have isolated primary mouse embryonic fibroblasts from mutants and introduced an epitope tagged form of SUMO. Preliminary results show that this form of SUMO can be efficiently conjugated to proteins and that these sumoylated proteins can be specifically immunoprecipitated. We are now developing proteomic approaches to determine their identities and allow an assessment of their potential roles in the mutant phenotype.

N4BP1 is a member of a small family of proteins containing a NYN motif, a domain predicted to have ribonuclease activity. N4BP1 undergoes polyubiquitination mediated by Nedd4, and also undergoes conjugation with SUMO, which in turn regulates N4BP1 ubiquitination and stability. Recent work has shown that N4BP1 also interacts with the related E3 ligase, ITCH, but is not a substrate for ITCH mediated ubiquitination. Rather, N4BP1 binding to ITCH, negatively regulates ITCH E3 activity directed toward its substrates, including the p53 related tumor suppressor proteins p73 and p63, as well as c-Jun. These results suggest that N4BP1 may have a role in regulating tumor progression and the response of cancer cells to chemotherapy.

A potential role in tumorigenesis has recently come to light for the second Nedd4 interaction partner under study, N4BP3. The locus has been identified as a common site for retroviral integration in mouse leukemogenesis. In addition, N4BP3 is now known to contain a so-called FEZ domain first identified in the tumor suppressor, LZTS1. Our recent studies have shown that Smurf1 and Smurf2, related E3 ubiquitin ligases implicated in the regulation of TGF-β and MEKK signaling, mediate N4BP3 polyubiquitination and proteasomal degradation, suggesting a function for N4BP3 in these pathways.

This page was last updated on 6/11/2008.