Ken Prehoda is an expert in molecular biology and molecular evolution. At the University of Oregon, he is a professor of chemistry and biochemistry. Ken studies how cells evolve over time and how cellular evolution can help find clues to diseases. He is particularly interested in how proteins work inside cells. Ken and his team identified an ancient mutation that contributed to evolution of multicellular animals.
Areas of Expertise (3)
Media Appearances (4)
Scientists dig up proteins from the past
Science News online
The influenza virus is a quick-change artist. In a few decades, its genome can evolve as much as animal genomes can over millions of years. That means that the viral proteins, including those that alert our bodies to an infection, constantly reinvent themselves, threatening our immune systems and frustrating vaccine developers.
Oregon Public Broadcasting online
Ken Prehoda, the director of the institute of Molecular Biology at the University of Oregon, tells us about the recent discovery about the evolutionary jump from single-cell to multi-cell organisms.
The rise of multicelled organisms
The Washington Post online
Ken Prehoda, a biochemist and director of the University of Oregon's Institute of Molecular Biology, discusses his research identifying the mutation that led to multicellular animals.
A mutation, a protein combo, and life went multicellular
Around the O online
All it took was one genetic mutation more than 600 million years ago. With that random act, a new protein function was born that helped our single-celled ancestor transition into an organized multicellular organism.
That's the scenario — done with some molecular time travel — that emerged from basic research in the lab of University of Oregon biochemist Ken Prehoda.
The mutation and a change it brought in protein interactions are detailed in a paper published in eLife, an open-access journal launched in 2012 with support of the Howard Hughes Medical Institute, the Max Planck Society and the Wellcome Trust.
During Drosophila development, neuroblasts divide to generate progeny with two different fates. One daughter cell self-renews to maintain the neuroblast pool, whereas the other differentiates to populate the central nervous system. The difference in fate arises from the asymmetric distribution of proteins that specify either self-renewal or differentiation, which is brought about by their polarization into separate apical and basal cortical domains during mitosis. Neuroblast symmetry breaking is regulated by numerous proteins, many of which have only recently been discovered. The atypical protein kinase C (aPKC) is a broad regulator of polarity that localizes to the neuroblast apical cortical region and directs the polarization of the basal domain. Recent work suggests that polarity can be explained in large part by the mechanisms that restrict aPKC activity to the apical domain and those that couple asymmetric aPKC activity to the polarization of downstream factors. Polarized aPKC activity is created by a network of regulatory molecules, including Bazooka/Par-3, Cdc42, and the tumor suppressor Lgl, which represses basal recruitment. Direct phosphorylation by aPKC leads to cortical release of basal domain factors, preventing them from occupying the apical domain. In this framework, neuroblast polarity arises from a complex system that orchestrates robust aPKC polarity, which in turn polarizes substrates by coupling phosphorylation to cortical release.
Membrane-associated guanylate kinases (MAGUKs) organize protein complexes at specific cellular sites by regulating interactions with their COOH-terminal guanylate kinase-like domains (GKs). Negative regulation of MAGUK GKs by an adjacent Src homology 3 domain (SH3) is critical for function, yet the mechanism is poorly understood. To gain insight into this process, we investigated SH3 regulation of the Discs large (Dlg) GK. Mutational analysis revealed that the binding site of the SH3-inhibited GK ligand GukHolder (GukH) is opposite the SH3 interacting surface, indicating that the SH3 does not directly occlude GukH binding. We screened for constitutively active SH3GK variants using yeast two-hybrid and a cell polarity/mitotic spindle orientation assay. Residues in both the SH3 and GK are required to maintain SH3GK inhibition, including those distant from both the SH3−GK and GK−GukH interaction sites. Activating mutations do not alter the ability of the SH3 and GK to interact in trans. On the basis of these observations, we propose that the SH3 modulates GK allostery to control its function.
Asymmetric cell division is intensely studied because it can generate cellular diversity as well as maintain stem cell populations. Asymmetric cell division requires mitotic spindle alignment with intrinsic or extrinsic polarity cues, but mechanistic detail of this process is lacking. Here, we develop a method to construct cortical polarity in a normally unpolarized cell line and use this method to characterize Partner of Inscuteable (Pins; LGN/AGS3 in mammals) -dependent spindle orientation. We identify a previously unrecognized evolutionarily conserved Pins domain (PinsLINKER) that requires Aurora-A phosphorylation to recruit Discs large (Dlg; PSD-95/hDlg in mammals) and promote partial spindle orientation. The well-characterized PinsTPR domain has no function alone, but placing the PinsTPR in cis to the PinsLINKER gives dynein-dependent precise spindle orientation. This “induced cortical polarity” assay is suitable for rapid identification of the proteins, domains, and amino acids regulating spindle orientation or cell polarity.
Background: Asymmetric cell divisions generate daughter cells with distinct fates by polarizing fate determinants into separate cortical domains. Atypical protein kinase C (aPKC) is an evolutionarily conserved regulator of cell polarity. In Drosophila neuroblasts, apically restricted aPKC is required for segregation of neuronal differentiation factors such as Numb and Miranda to the basal cortical domain. Whereas Numb is polarized by direct aPKC phosphorylation, Miranda asymmetry is thought to occur via a complicated cascade of repressive interactions (aPKC −| Lgl −| myosin II −| Miranda).
Results: Here we provide biochemical, cellular, and genetic data showing that aPKC directly phosphorylates Miranda to exclude it from the cortex and that Lgl antagonizes this activity. Miranda is phosphorylated by aPKC at several sites in its cortical localization domain and phosphorylation is necessary and sufficient for cortical displacement, suggesting that the repressive-cascade model is incorrect. In investigating key results that led to this model, we found that Y-27632, a Rho kinase inhibitor used to implicate myosin II, efficiently inhibits aPKC. Lgl3A, a nonphosphorylatable Lgl variant used to implicate Lgl in this process, inhibits the formation of apical aPKC crescents in neuroblasts. Furthermore, Lgl directly inhibits aPKC kinase activity.
Conclusions: Miranda polarization during neuroblast asymmetric cell division occurs by displacement from the apical cortex by direct aPKC phosphorylation. Rather than mediating Miranda cortical displacement, Lgl instead promotes aPKC asymmetry by regulating its activity. The role of myosin II in neuroblast polarization, if any, is unknown.
Membrane-associated guanylate kinases (MAGUKs) regulate the formation and function of molecular assemblies at specialized regions of the membrane. Allosteric regulation of an intramolecular interaction between the Src homology 3 (SH3) and guanylate kinase (GK) domains of MAGUKs is thought to play a central role in regulating MAGUK function. Here we show that a mutant of the Drosophila MAGUK Discs large (Dlg), dlgsw, encodes a form of Dlg that disrupts the intramolecular association while leaving the SH3 and GK domains intact, providing an excellent model system to assess the role of the SH3-GK intramolecular interaction in MAGUK function. Analysis of asymmetric cell division of maternal-zygotic dlgsw embryonic neuroblasts demonstrates that the intramolecular interaction is not required for Dlg localization but is necessary for cell fate determinant segregation to the basal cortex and mitotic spindle alignment with the cortical polarity axis. These defects ultimately result in improper patterning of the embryonic central nervous system. Furthermore, we demonstrate that the sw mutation of Dlg results in unregulated complex assembly as assessed by GukHolder association with the SH3-GK versus PDZ-SH3-GK modules of Dlgsw. From these studies, we conclude that allosteric regulation of the SH3-GK intramolecular interaction is required for regulation of MAGUK function in asymmetric cell division, possibly through regulation of complex assembly.