Soybean nodule autoregulation receptor kinase phosphorylates two kinase-associated protein phosphatases in vitro.
The NARK (nodule autoregulation receptor kinase) gene, a negative regulator of cell proliferation in nodule primordia in several legumes, encodes a receptor kinase that consists of an extracellular leucine-rich repeat and an intracellular serine/threonine protein kinase domain. The putative catalytic domain of NARK was expressed and purified as a maltose-binding or a glutathione S-transferase fusion protein in Escherichia coli. The recombinant NARK proteins showed autophosphorylation activity in vitro. Several regions of the NARK kinase domain were shown by mass spectrometry to possess phosphoresidues. The kinase-inactive protein K724E failed to autophosphorylate, as did three other proteins corresponding to phenotypically detected mutants defective in whole plant autoregulation of nodulation. A wild-type NARK fusion protein transphosphorylated a kinase-inactive mutant NARK fusion protein, suggesting that it is capable of intermolecular autophosphorylation in vitro. In addition, Ser-861 and Thr-963 in the NARK kinase catalytic domain were identified as phosphorylation sites through site-directed mutagenesis. The genes coding for the kinase-associated protein phosphatases KAPP1 and KAPP2, two putative interacting components of NARK, were isolated. NARK kinase domain phosphorylated recombinant KAPP proteins in vitro. Autophosphorylated NARK kinase domain was, in turn, dephosphorylated by both KAPP1 and KAPP2. Our results suggest a model for signal transduction involving NARK in the control of nodule development.
Miyahara
Akira
A
Australian Research Council Centre of Excellence for Integrative Legume Research, St. Lucia, Brisbane, Queensland 4072, Australia.
Hirani
Tripty A
TA
Australian Research Council Centre of Excellence for Integrative Legume Research, St. Lucia, Brisbane, Queensland 4072, Australia.
Oakes
Marie
M
Australian Research Council Centre of Excellence for Integrative Legume Research, Research School of Biological Sciences, Australian National University, Canberra, Australian Capital Territory 2601, Australia.
Kereszt
Attila
A
Australian Research Council Centre of Excellence for Integrative Legume Research, St. Lucia, Brisbane, Queensland 4072, Australia.
Kobe
Bostjan
B
School of Microbial and Molecular Sciences and Institute for Molecular Bioscience, University of Queensland, St. Lucia, Brisbane, Queensland 4072, Australia and the.
Djordjevic
Michael A
MA
Australian Research Council Centre of Excellence for Integrative Legume Research, Research School of Biological Sciences, Australian National University, Canberra, Australian Capital Territory 2601, Australia.
Gresshoff
Peter M
PM
Australian Research Council Centre of Excellence for Integrative Legume Research, St. Lucia, Brisbane, Queensland 4072, Australia. Electronic address: p.gresshoff@uq.edu.au.
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000378:metabolism; D020134:Catalytic Domain; D049109:Cell Proliferation; D004926:Escherichia coli / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013025:Glycine max / Q000378:metabolism*
D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D010770:Phosphotransferases; D017346:Protein Serine-Threonine Kinases; D005978:Glutathione