title: Arabidopsis receptor of activated C kinase1 phosphorylation by WITH NO LYSINE8 KINASE.
abstract: Receptor of activated C kinase1 (RACK1) is a versatile scaffold protein that binds to numerous proteins to regulate diverse cellular pathways in mammals. In Arabidopsis (Arabidopsis thaliana), RACK1 has been shown to regulate plant hormone signaling, stress responses, and multiple processes of growth and development. However, little is known about the molecular mechanism underlying these regulations. Here, we show that an atypical serine (Ser)/threonine (Thr) protein kinase, WITH NO LYSINE8 (WNK8), phosphorylates RACK1. WNK8 physically interacted with and phosphorylated RACK1 proteins at two residues: Ser-122 and Thr-162. Genetic epistasis analysis of rack1 wnk8 double mutants indicated that RACK1 acts downstream of WNK8 in the glucose responsiveness and flowering pathways. The phosphorylation-dead form, RACK1A(S122A/T162A), but not the phosphomimetic form, RACK1A(S122D/T162E), rescued the rack1a null mutant, implying that phosphorylation at Ser-122 and Thr-162 negatively regulates RACK1A function. The transcript of RACK1A(S122D/T162E) accumulated at similar levels as those of RACK1(S122A/T162A). However, although the steady-state level of the RACK1A(S122A/T162A) protein was similar to wild-type RACK1A protein, the RACK1A(S122D/T162E) protein was nearly undetectable, suggesting that phosphorylation affects the stability of RACK1A proteins. Taken together, these results suggest that RACK1 is phosphorylated by WNK8 and that phosphorylation negatively regulates RACK1 function by influencing its protein stability.
authors.affiliation: Departments of Biology (D.U., A.M.J.) andPharmacology (A.M.J.), University of North Carolina, Chapel Hill, North Carolina 27599;Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831 (O.C., X.W., J.-G.C.); andKey Laboratory of Molecular Epigenetics of Ministry of Education and Institute of Genetics and Cytology, Northeast Normal University, Changchun 130024, China (X.W.).
authors.forename: Daisuke
authors.initials: D
authors.lastname: Urano
authors.affiliation: Departments of Biology (D.U., A.M.J.) andPharmacology (A.M.J.), University of North Carolina, Chapel Hill, North Carolina 27599;Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831 (O.C., X.W., J.-G.C.); andKey Laboratory of Molecular Epigenetics of Ministry of Education and Institute of Genetics and Cytology, Northeast Normal University, Changchun 130024, China (X.W.).
authors.forename: Olaf
authors.initials: O
authors.lastname: Czarnecki
authors.affiliation: Departments of Biology (D.U., A.M.J.) andPharmacology (A.M.J.), University of North Carolina, Chapel Hill, North Carolina 27599;Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831 (O.C., X.W., J.-G.C.); andKey Laboratory of Molecular Epigenetics of Ministry of Education and Institute of Genetics and Cytology, Northeast Normal University, Changchun 130024, China (X.W.).
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Wang
authors.affiliation: Departments of Biology (D.U., A.M.J.) andPharmacology (A.M.J.), University of North Carolina, Chapel Hill, North Carolina 27599;Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831 (O.C., X.W., J.-G.C.); andKey Laboratory of Molecular Epigenetics of Ministry of Education and Institute of Genetics and Cytology, Northeast Normal University, Changchun 130024, China (X.W.) alan_jones@unc.edu chenj@ornl.gov.
authors.forename: Alan M
authors.identifier: http://orcid.org/0000-0002-2365-6462
authors.initials: AM
authors.lastname: Jones
authors.affiliation: Departments of Biology (D.U., A.M.J.) andPharmacology (A.M.J.), University of North Carolina, Chapel Hill, North Carolina 27599;Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831 (O.C., X.W., J.-G.C.); andKey Laboratory of Molecular Epigenetics of Ministry of Education and Institute of Genetics and Cytology, Northeast Normal University, Changchun 130024, China (X.W.) alan_jones@unc.edu chenj@ornl.gov.
authors.forename: Jin-Gui
authors.initials: JG
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000378:metabolism*; D005816:Genetic Complementation Test; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*
chemical_list: D029681:Arabidopsis Proteins; C515163:RACK1 protein, Arabidopsis; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface; D010768:Phosphoserine; D017346:Protein Serine-Threonine Kinases; C508913:WNK8 protein, Arabidopsis