Analysis of the Complexity of Protein Kinases within the Phloem Sieve Tube System: CHARACTERIZATION OF CUCURBITA MAXIMA CALMODULIN-LIKE DOMAIN PROTEIN KINASE 1

Byung-Chun Yoo,Jung-Youn Lee,William J Lucas

doi:10.1074/jbc.m200382200

Byung-Chun Yoo, Jung-Youn Lee + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m200382200

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Abstract

In angiosperms, functional, mature sieve elements lack nuclei, vacuoles, ribosomes, and most of the endomembrane network. In this study, the complexity, number, and nature of protein kinases within the phloem sap of Cucurbita maxima were investigated to test the hypothesis that the enucleate sieve tube system utilizes a simplified signal transduction network. Supporting evidence was obtained in that only five putative protein kinases (three calcium-independent and two calcium-dependent protein kinases) were detected within the phloem sap extracted from stem tissues. Biochemical methods were used to purify one such calcium-dependent protein kinase. The gene for this C. maxima calmodulin-like domain protein kinase 1 (CmCPK1), was cloned using peptide microsequences. A combination of mass spectrometry, peptide fingerprinting, and amino-terminal sequencing established that, in the phloem sap, CmCPK1 exists as an amino-terminally cleaved protein. A second highly homologous isoform, CmCPK2, was identified, but although transcripts could be detected in the companion cells, peptide fingerprint analysis suggested that CmCPK2 does not enter the phloem sap. Potential substrates for CmCPK1, within the phloem sap, were also detected using an on-membrane phosphorylation assay. Entry of CmCPK1 into sieve elements via plasmodesmata and the potential roles played by these phloem protein kinases are discussed.

Highlights

In angiosperms, functional, mature sieve elements lack nuclei, vacuoles, ribosomes, and most of the endomembrane network
Equal amounts of protein extracted from pumpkin phloem sap and vascular tissue were first resolved by SDS-PAGE (Fig. 1A), followed by further denaturation with 6 M guanidine-HCl, and renatured in the gel
The limited number of proteins detected within the phloem sap that were capable of autophosphorylation (Figs. 1 and 2), thereby likely representing potential protein kinases, is consistent with the hypothesis that functional, enucleate sieve element (SE) utilize a simplified signal transduction network

Summary

Introduction

Functional, mature sieve elements lack nuclei, vacuoles, ribosomes, and most of the endomembrane network. The complexity, number, and nature of protein kinases within the phloem sap of Cucurbita maxima were investigated to test the hypothesis that the enucleate sieve tube system utilizes a simplified signal transduction network. Equal amounts of protein extracted from pumpkin phloem sap and vascular tissue were first resolved by SDS-PAGE (Fig. 1A), followed by further denaturation with 6 M guanidine-HCl, and renatured in the gel.

Results

Conclusion