PROTEIN-PROTEIN INTERACTIONS OF TANDEM AFFINITY PURIFICATION (TAP)-TAGGED PROTEIN KINASES IN RICE

Authors: ROHILA, JAI - UNIV OF NEBRASKA; CHEN, MEI - UNIV OF NEBRASKA; CHEN, SHUO - UNIV OF NEBRASKA; CHEN, JOHANN - UNIV OF CALIFORNIA; CERNY, RONALD - UNIV OF NEBRASKA; Dardick, Christopher - Chris; CANLAS, PATRICK - UNIV OF CALIFORNIA; XU, XIA - UNIV OF CALIFORNIA; GRIBSKOV, MICHAEL - PURDUE UNIV; KANRAR, SIDDHARTHA - UNIV OF CALIFORNIA; ZHU, JIAN-KANG - UNIV OF CALIFORNIA; RONALD, PAMELA - UNIV OF CALIFORNIA; FROMM, MICHAEL - UNIV OF NEBRASKA

Submitted to: Plant Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/12/2006
Publication Date: 7/26/2006
Citation: Rohila, J.S., Chen, M., Chen, S., Chen, J., Cerny, R., Dardick, C.D., Canlas, P., Xu, X., Gribskov, M., Kanrar, S., Zhu, J., Ronald, P., Fromm, M. 2006. Protein-protein interactions of tandem affinity purification (tap)-tagged protein kinases in rice. Plant Journal. 46:1-13.

Interpretive Summary: At the molecular level, cell signaling networks allow plants to respond to environmental changes with developmental, biochemical, and physiological adaptations. These signaling networks are typically controlled by a protein modification termed phosphorylation and the enzymes responsible, protein kinases and phosphatases, make up about 5% of the genes in plants. Protein kinases and phosphatases are critical components of most plant signaling pathways including cold, drought and salt tolerance, disease resistance, leaf abscission, fruit development, light perception, developmental signaling, regulation of carbon metabolism, and cell cycle regulation. Kinases are often found as constituents of signaling complexes along with multiple regulatory proteins and are held together by anchoring or adaptor proteins. Discovering the protein components of these signaling complexes is critical to understanding how cell signaling networks function and potentially finding new ways to control them. New technologies enable scientists to identify the protein components in complex mixtures. These include advances in mass spectrometry (MS) and tandem affinity purification (TAP) of tagged protein complexes from yeast, insect and human cells, and plants. In order to learn more about protein kinase signaling networks, we have initiated a project to TAP-tag rice protein kinases one-by-one, express them in transgenic rice plants and identify which rice proteins are associated with each kinase. We report here the analysis of 41 TAP-tagged rice protein kinases purified from rice plants. We found 95% of the TAP-tagged rice protein kinases can be purified and 56% of these have been isolated as complexes with one or more interacting proteins. The validity of some of the interacting proteins in the isolated protein complexes is supported by evidence from interactions of similar proteins in other organisms. The results of this work reveal important aspects of cell signaling networks in plants and provide new gene targets for manipulation of numerous plant qualities including growth and development, stress tolerance, and pathogen resistance.

Technical Abstract: Forty-one rice cDNAs encoding protein kinases were fused to the tandem affinity purification (TAP) tag and expressed in transgenic rice plants. The TAP-tagged kinases and interacting proteins were purified from the T1 progeny of the transgenic rice plants and identified by mass spectrometry. Ninety-five percent of the TAP-tagged kinases were recovered. Fifty-six percent of the TAP-tagged kinases were found to interact with other rice proteins. A number of these interactions were consistent with known protein complexes found in other species, validating the TAP-tag method in rice plants. Phosphorylation sites were identified on four of the kinases that interacted with either 14-3-3 proteins or cyclins.