ESTELLE M. HRABAK Associate Professor of Plant Biology and Genetics Ph.D., University of Wisconsin, Madison, 1992 estelle.hrabak@unh.edu

The Hrabak lab uses molecular, genetic and biochemical approaches to study enzymes involved in signal transduction processes in plants. We focus on the model plants Arabidopsis thaliana and Physcomitrella patens.

1. Calcium-dependent Protein Kinases

Calcium ions are important second messengers during plant growth and development. However, many of the molecules that function as calcium sensors, as well as the downstream signal transduction pathways, are poorly understood. Calcium-dependent protein kinases (CDPK), a family of serine-threonine kinases found primarily in the plant kingdom, are likely to function as sensor molecules in calcium-mediated signaling pathways. The genome of the plant Arabidopsis thaliana contains 34 genes predicted to encode CDPKs. All CDPK proteins have a four-domain structure with variable, kinase catalytic, autoregulatory, and calmodulin-like domains. Calcium binding to the calmodulin-like domain activates the enzyme by inducing a conformational change that relieves inhibition by the autoregulatory domain. CDPK enzyme activity is found in membrane and soluble fractions and potential CDPK substrates include both membrane proteins (such as the KAT1 potassium channel and the H⁺-ATPase) and soluble proteins (such as sucrose phosphate synthase and nitrate reductase).

The long-term goal of our research is to understand the functions of CDPKs in plants. We are using multiple experimental approaches to achieve this goal. Knowledge of the cellular and subcellular locations of proteins can provide clues to their functions. We have expressed CDPK-GUS ß-glucuronidase) fusion proteins in Arabidopsis under control of the native CDPK promoter to determine cellular expression patterns. These experiments showed that many CDPKs have distinct expression patterns during Arabidopsis development. These results are being confirmed by RT-PCR. CDPK-GFP (green fluorescent protein) fusions are being used to examine subcellular localization. As expected, many CDPKs are wholly or partially membrane localized. We have shown that membrane binding is facilitated by myristoylation and possibly palmitoylation of CDPKs. Identification of proteins that interact with calcium-dependent protein kinases may identify kinase substrates or regulatory proteins. We are using yeast two-hybrid analysis and immunoprecipitation to identify CDPK interactors.

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2. Protein Phosphatases

Protein phosphatases reverse the action of protein kinases by removing phosphate groups from amino acids on their substrate proteins. Plants have four major groups of protein phosphatases: the PP1, PP2A, and PP2C groups are serine/threonine phosphatases, while PTPs are protein tyrosine phosphatases. We are concentrating on the PP2A group. These heterotrimeric enzymes contain a scaffolding subunit (A), a catalytic subunit (C), and a subunit involved in targeting and specificity (B). We have identified T-DNA insertion mutants in the many of the genes encoding the A, B, and C subunits of Arabidopsis PP2A and are currently characterizing the phenotypes associated with these mutations.

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Representative Publications

Hrabak, E.M., C.W.M. Chan, M. Gribskov, J.F. Harper, J.H. Choi, N. Halford, J. Kudla, S. Luan, H.G. Nimmo, M.R. Sussman, M. Thomas, K. Walker-Simmons, J.-K. Zhu, and A.C. Harmon. 2003. The Arabidopsis CDPK-SnRK superfamily of protein kinases. Plant Physiol. 132:666-680

Lu, S.X. and E.M. Hrabak. 2002. An Arabidopsis calcium-dependent protein kinase is associated with the endoplasmic reticulum. Plant Physiol. 128:1008-1021

Hrabak, E.M. 2000. Calcium-dependent protein kinases and their relatives. Adv. Bot. Res. 32:185-223

Hrabak, E.M., L.J. Dickmann, J.S. Satterlee, and M.R. Sussman. 1996. Characterization of eight new members of the calmodulin-like domain protein kinase gene family of Arabidopsis thaliana. Plant Mol. Biol. 31:405-412