Office: BMSB 803
Phone: (405) 271-2227 ext. 61219
Fax: (405) 271-3092
940 S. L. Young Blvd., BMSB 803
Oklahoma City, OK 73104
PhD, Kyoto University, Japan, 1977
Molecular mechanism of visual excitation; role of protein phosphorylation in neuronal function; molecular biology of cellular regulation in neurons.
The long-term goal of my laboratory's research is to elucidate the role of protein phosphorylation in the excitation and adaptation processes of both vertebrate and invertebrate photoreceptors. In order to achieve this goal we have been developing microanalytical techniques for proteins to study subtle changes in the amino acid side chains caused by post-translational modification such as protein phosphorylation by using modern mass spectrometry. In the last several years, technical developments in mass spectrometry enabled us to ionize non-volatile biomolecules including proteins, peptides, and nucleic acids. Mass spectrometers capable of ionizing biomolecules became commercially available just recently. We have developed techniques to interface two-dimensional (2-D) gel electrophoresis to such modern mass spectrometry. This involves a streamlined procedure consisting of 2-D gel, in-gel digestion, micro-bore HPLC, and HPLC interfaced with an electrospray tandem quadrupole mass spectrometer (ESIMS). In addition to HPLC-ESIMS we recently started using matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOFMS) in our protocol. Using such modern mass spectrometry combined with sophisticated biochemical techniques such as 2-D gel electrophoresis, in-gel digestion, micro-bore HPLC, and Edman degradation, we are focusing our effort on two major subjects: 1) Quantitative use of mass spectrometry especially for phosphopeptides, and 2) microanalysis of proteins and protein cataloging using both HPLC-ESIMS and MALDI-TOFMS. The five on-going projects are described below.
(1) Protein phosphorylation cascades in the compound eyes of Drosophila
Protein phosphorylation plays crucial roles in cellular signaling. Using Drosophila melanogaster as a model system, we have been studying phosphorylation and dephosphorylation of an arrestin homolog, phosrestin I, that we had discovered and characterized in the compound eyes of the fly. In the past several years our primary concern has been the cascade responsible for the phosphorylation of phosrestin I in vivo. In fly photoreceptors, polyphosphoinositide-specific phospholipase C (PI-PLC), instead of cGMP phosphodiesterase, is activated through a photoreceptor-specific G protein. The activation of PI-PLC potentially activates two protein phosphorylation cascades, protein kinase C (PKC) and calcium/calmodulin-dependent protein kinase (CaMK). Our works unequivocally demonstrated that 1) phosrestin I undergoes the earliest phosphorylation induced by light, 2) the phosphorylation of phosrestin I is downstream of both the excitation of rhodopsin and the activation of PI-PLC, 3) the CaMK pathway rather than the PKC pathway is responsible for the phosphorylation of phosrestin I in vivo, 4) phosrestin I becomes phosphorylated at the Ser366 in vivo, and 5) the CaMK responsible for the phosphorylation of phosrestin I belongs to Type II (CaMKII). We are currently focusing our effort to establish a method to quantify the phosphorylation state of phosrestin I by MALDI-TOFMS. This method will allow us to follow the phosphorylation of phosrestin I and other phosphoproteins in detail without using any radiolabels and at a miniscule scale. Our current focus is on the role of phosphorylation of phosrestin I at the molecular level, which can be addressed by questions such as "How does the phosphorylation of phosrestin I affect its binding to rhodopsin?" or "How does the phosphorylation of phosrestin I affect the activation of PI-PLC by the rhodopsin-activated G protein cascade?" The elucidation of the regulatory mechanism of phosrestin I will not only reveal the molecular mechanisms of fly vision, but also will lead us to a better understanding of PI-PLC regulation in other types of cells. Since the activation of PI-PLC is one of the main events involved in cellular signaling, the achievement of this project goal will significantly contribute to our knowledge in signal transduction in general.
(2) Regulation of InaD protein, a member of PDZ family, by multiple phosphorylation
We recently discovered that the Drosophila 80K protein is the InaD gene product, a PDZ family protein by peptide mass fingerprinting. Available evidence suggests that the 80K(InaD) protein adjusts photoreceptor responsiveness by assembling/disassembling components involved in the photoreceptor transduction in fly eyes. The phosphorylation states of 80K(InaD) depend on the intensity and/or duration of light stimuli. We postulate that the 80K(InaD) protein functions as a molecular switch adjusting the signaling cascade through phosphorylation at multiple sites. Our effort is directed toward deciphering such switching mechanism by the combination of a genetic approach and a biochemical approach using modern mass spectrometry.
(3) Protein phosphorylation cascades in vertebrate photoreceptors
In the past several years our group has been collaborating with Dr. Akio Yamazaki's group at Wayne State University in Detroit in order to elucidate the role of phosphorylation and ADP-ribosylation of the gamma subunit (Pg ) of cGMP phosphodiesterase (PDE) in bovine and frog photoreceptors. Since PDE is the key enzyme that is activated by the rhodopsin-activated transducin cascade and since Pg is an inhibitory subunit on PDE, the effect of post-translational modification of Pg on the PDE activity is a crucial factor in the regulation of visual transduction. Our experimental results indicate that the phosphorylation and ADP-ribosylation of Pg by endogenous enzymes enhances the inhibitory action of Pg in vitro. The results suggest that phosphorylation and ADP-ribosylation of Pg can participate in the shut off mechanism of photoreceptor excitation. Our current concern is to prove that these modifications also take place in vivo by microanalytical techniques using mass spectrometry.
(4) Catalog of vertebrate retinal proteins
Although the major pathway of excitation mechanism in vertebrate photoreceptors is well established, the mechanisms that regulate adaptation/desensitization remain obscure. Presumably, the major players in photoreceptor-specific functions are present specifically in the photoreceptor cells. Therefore, a catalog of these proteins will provide a useful tool for vision researchers. We have developed a novel method for isolating the photoreceptor cell monolayer (PCL) from bovine retina that minimizes loss of soluble proteins. Microanalytical techniques including 2-D gel, in-gel digestion, micro-bore HPLC, Edman degradation, and mass spectrometry are utilized for the generation of amino acid sequence data. These data permit both the identification of virtually any protein detectable on a 2-D gel, and also enable the corresponding cDNA clone to be selected. Our goal is 1) to expand the catalog of photoreceptor proteins and proteins expressed in other types of retinal cells, and 2) after identifying proteins, which have been reported to be phosphorylated in vitro, to confirm and to identify the phosphorylation site(s) in vivo.
(5) Development of microscale biochemical analysis by mass spectrometry
We are interested in developing technologies to utilize both HPLC-ESIMS and MALDI-TOFMS for the microanalysis of proteins, peptides, DNAs, carbohydrates, and other biomedical-related molecules. This is also one of my missions as Director of NSF EPSCoR Oklahoma Biotechnology Network Laser Mass Spectrometry Facility. I would like to pursue this in the context of my on-going projects as well as in the context of general interest. One major direction is to develop a general method to quantify phosphopeptide in the mixture with its non-phosphorylated form. Mass spectrometry, in general, tends to be non-quantitative because of the difference of ionization efficiencies for different molecules. However, we could overcome this difficulty by carefully running standard samples and calibrating the measurement. I believe that in the future biochemists will be using mass spectrometry in their routine experiments. Our long-term goal on this line of the project is to develop such routine protocols for microanalysis using mass spectrometry.
- ·Matsukura, T., Inaba, C., Weygant, E.A., Kitamura, D., Janknecht, R., Matsumoto, H., Hyink, D.P., Obara, T. 2019. Extracellular vesicles from human bone marrow mesenchymal stem cells repair organ damage caused by cadmium poisoning in a medaka model. Physiol. Rep. https://doi.org/10.14814/phy2.14172.
- Mecklenburg, K.#, Takemori, N.#, Komori, N., Chu, B., Hardie, R., Matsumoto, H.*, and O’Tousa, J.* 2010. Retinophilin is a NINAC- and light-regulated phosphoprotein required for suppression of dark noise in Drosophila photoreceptors. Journal of Neuroscience, 30(4): 1238-1249, PMID: 20107052. (# equal contribution; * co-corresponding authors)
- Matsumoto, H., Kurien, B., Takagi, Y., Kahn, E.S., Kinumi, T., Komori, N., Yamada, T., Hayashi, F., Isono, K., Pak, W.L., Jackson, K.W., and Tobin, S.L. 1994. Phosrestin I undergoes the earliest light-induced phosphorylation by a calcium/calmodulin-dependent protein kinase in Drosophila photoreceptors. Neuron 12: 997-1010. PMID: 8185954
- Yamada, T., Takeuchi, Y., Komori, N., Kobayashi, H., Sakai, Y., Hotta, Y., and Matsumoto, H. 1990. A 49-kilodalton Phosphoprotein in the Drosophila photoreceptor is an Arrestin-Homolog. Science 248: 483-486. PMID: 2158671
- Matsumoto, H., Isono, K., Pye, Q.N., and Pak, W.L. 1987. Gene encoding cytoskeletal proteins in Drosophila rhabdomeres. Proc. Natl. Acad. Sci. USA 84: 985-989.
- Matsumoto, H., and Pak, W.L. 1984. Light-induced phosphorylation of retina-specific polypeptides of Drosophila in vivo. Science 223: 184-186. PMID: 6419348
- Matsumoto, H., O'Tousa, J., and Pak, W.L. 1982. Light-induced modification of Drosophila retinal polypeptides in vivo. Science 217: 839-841.
- Matsumoto, H., and Yoshizawa, T. 1975. Existence of a b-ionone ring-binding site in the rhodopsin molecule. Nature 258: 523-526.