STN8 Protein Kinase in Arabidopsis thaliana Is Specific in Phosphorylation of Photosystem II Core Proteins

Abstract

light-exposed leaves of two Arabidopsis lines with T-DNA insertions in the stn8 gene was found significantly reduced in the assays with anti-phosphothreonine antibodies. Protein phosphorylation in each of the mutants was quantified comparatively to the wild type by mass spectrometric analyses of phosphopeptides released from the photosynthetic membranes and differentially labeled with stable isotopes. The lack of STN8 caused 50 -60% reduction in D1 and D2 phosphorylation, but did not change the phosphorylation level of two peptides that could correspond to light-harvesting proteins encoded by seven different genes in Arabidopsis. Phosphorylation of the PsbH protein at Thr-4 was completely abolished in the plants lacking STN8. Phosphorylation of Thr-4 in the wild type required both light and prior phosphorylation at Thr-2, indicating that STN8 is a light-activated kinase that phosphorylates Thr-4 only after another kinase phosphorylates Thr-2. Analysis of the STN8 catalytic domain suggests that selectivity of STN8 in phosphorylation of the very N-terminal residues in D1, D2, and CP43, and Thr-4 in PsbH pre-phosphorylated at Thr-2 may be explained by the long loops obstructing entrance into the kinase active site and seven additional basic residues in the vicinity of the catalytic site, as compared with the homologous STN7 kinase responsible for phosphorylation of light-harvesting proteins.Reversible protein phosphorylation is a key molecular mechanism regulating all aspects of physiology and development in eukaryotic cells. The genome sequencing of Arabidopsis thaliana has uncovered more than 1160 genes encoding for the protein kinases and phosphatases in this model plant (1), highlighting a new challenge in revealing of the substrates and functions for these catalysts of reversible protein phosphorylation. The task of identification of in vivo substrate specificities for individual protein kinases and phosphatases in Arabidopsis thaliana has recently became feasible because of two groundbreaking developments. First, the plant lines with knockouts of individual genes became public and commercially available (2). Second, new mass spectrometrybased analytical techniques permitted identification and mapping of in vivo phosphorylation sites in numerous proteins (as reviewed in Ref.3). For the first time these technical advancements provide the possibility of unraveling the complex protein phosphorylation network in plant photosynthetic membranes, which regulates the adaptation of the photosynthetic apparatus and efficient energy utilization in response to light quality and intensity, ambient temperature, circadian rhythm, nutrient deficiency, and other stresses (4 -6).The major proteins undergoing reversible phosphorylation in the photosynthetic thylakoid membranes belong to photosystem II (PSII) 3 and its light-harvesting antennae proteins LHCII (6 -11). Phosphorylation status of these proteins is differentially regulated by light (12), redox state of the membrane electron carriers (13-15), the ferredoxin-thioredoxi...

Protein kinases and phosphatases involved in the acclimation of the photosynthetic apparatus to a changing light environment

“…Together, they form a duo of kinases that is conserved in land plants where the orthologues of Stt7 and Stl1 are called STN7 and STN8, respectively [29]. Although these two kinases appear to have distinct substrates and functions with Stt7/STN7 required for LHCII phosphorylation and state transitions [27,29] and Stl1/STN8 for PSII core protein phosphorylation [30,31], there is some overlap between these two kinases as seen by the comparison of the protein phosphorylation patterns of stn7 and stn8 with that of the stn7-stn8 double mutant. While several proteins are still phosphorylated both in the stn7 or stn8 mutant, most of these phosphorylations are lost in the double mutant.…”

“…In this way, two allelic mutants were identified which were unable to dephosphorylate LHCII upon transfer from state 2 to state 1. The gene inactivated in these mutants encodes a PP2C phosphatase [27,[29][30][31]. STN7 is also involved in retrograde signalling (LTR) [30].…”

“…The principal substrates of STN8 are the PSII core proteins D1, D2, CP43 and PsbH, and the Ca 2þ -sensitive thylakoid phosphoprotein, CAS [30,31].…”

Natural variation in phosphorylation of photosystem II proteins in Arabidopsis thaliana : is it caused by genetic variation in the STN kinases?

“…Analysis of the protein phosphorylation profiles of the stn7 and stn8 mutants of Arabidopsis showed residual phosphorylation of the LHCII and PSII core proteins, respectively. However, this residual phosphorylation was undetectable in the stn7 Â stn8 double mutant, indicating some substrate overlap between the STN7 and STN8 kinases [8,15].…”

“…PSII core protein phosphorylation in general, and D1 phosphorylation in particular, has been suggested to facilitate disassembly of photoinactivated PSII complexes and is thought to play a role in the regulation of PSII repair [7]. The kinase involved in PSII core protein phosphorylation was identified in Arabidopsis and named STN8 [8,9].…”