Kinase-associated Protein Phosphatase Research Articles

The role of forkhead-associated (FHA)-domain proteins in plant biology.

The forkhead-associated (FHA) domain, a well-characterized small protein module that mediates protein-protein interactions by targeting motifs containing phosphothreonine, is present in many regulatory molecules like protein kinase, phosphatases, transcription factors, and other functional proteins. FHA-domain containing proteins in yeast and human are involved in a large variety of cellular processes such as DNA repair, cell cycle arrest, or pre-mRNA processing. Since the first FHA-domain protein, kinase-associated protein phosphatase (KAPP) was found in plants, the interest in plant FHA-containing proteins has increased dramatically, mainly due to the important role of FHA domain-containing proteins in plant growth and development. In this review, we provide a comprehensive overview of the fundamental properties of FHA domain-containing proteins in plants, and systematically summarized and analyzed the research progress of proteins containing the FHA domain in plants. We also emphasized that AT5G47790 and its homologs may play an important role as the regulatory subunit of protein phosphatase 1 (PP1) in plants.

The Arabidopsis kinase-associated protein phosphatase KAPP, interacting with protein kinases SnRK2.2/2.3/2.6, negatively regulates abscisic acid signaling.

The kinase-associated protein phosphatase, KAPP, is negatively involved in abscisic acid (ABA) signaling. KAPP interacts physically with SnRK2.2, SnRK2.3 and SnRK2.6, and functionally acts upstream of SnRK2.2 and SnRK2.3. The kinase-associated protein phosphatase (KAPP) has been reported to be involved in the regulation of many developmental and signaling events, but it remains unknown whether KAPP is involved in ABA signaling. Here, we report that KAPP is negatively involved in ABA-mediated seed germination and early seedling growth in Arabidopsis thaliana. The two loss-of-function mutants of KAPP, kapp-1 and kapp-2, exhibit increased ABA sensitivity in ABA-induced seed germination inhibition and post-germination growth arrest. The three closely-related protein kinase, (SNF1)-related protein kinase SnRK2.2, SnRK2.3 and SnRK2.6, which play critical roles in ABA signaling, interact and co-localize with KAPP. Genetic evidence showed that the ABA-hypersensitive phenotypes caused by KAPP mutation were suppressed by the double mutation of SnRK2.2 and SnRK2.3, indicating that KAPP functions upstream of SnRK2.2 and SnRK2.3 in ABA signaling. RNA-sequencing analysis revealed that KAPP mutation affects expression of multiple ABA-responsive genes. These results demonstrated that KAPP is negatively involved in plant response to ABA, which help to understand the complicated ABA signaling mechanism.

Progress on deciphering the molecular aspects of cell-to-cell communication in Brassica self-incompatibility response.

The sporophytic system of self-incompatibility is a widespread genetic phenomenon in plant species, promoting out-breeding and maintaining genetic diversity. This phenomenon is of commercial importance in hybrid breeding of Brassicaceae crops and is controlled by single S locus with multiple S haplotypes. The molecular genetic studies of Brassica 'S' locus has revealed the presence of three tightly linked loci viz. S-receptor kinase (SRK), S-locus cysteine-rich protein/S-locus protein 11 (SCR/SP11), and S-locus glycoprotein (SLG). On self-pollination, the allele-specific ligand-receptor interaction activates signal transduction in stigma papilla cells and leads to rejection of pollen tube on stigmatic surface. In addition, arm-repeat-containing protein 1 (ARC1), M-locus protein kinase (MLPK), kinase-associated protein phosphatase (KAPP), exocyst complex subunit (Exo70A1) etc. has been identified in Brassica crops and plays a key role in self-incompatibility signaling pathway. Furthermore, the cytoplasmic calcium (Ca2+) influx in papilla cells also mediates self-incompatibility response in Brassicaceae, but how this cytoplasmic Ca2+ influx triggers signal transduction to inhibit pollen hydration is still obscure. There are many other signaling components which are not well characterized yet. Much progress has been made in elucidating the downstream multiple pathways of Brassica self-incompatibility response. Hence, in this review, we have made an effort to describe the recent advances made on understanding the molecular aspects of genetic mechanism of self-incompatibility in Brassicaceae.

GRP-3 and KAPP, encoding interactors of WAK1, negatively affect defense responses induced by oligogalacturonides and local response to wounding.

Conserved microbe-associated molecular patterns (MAMPs) and damage-associated molecular patterns (DAMPs) act as danger signals to activate the plant immune response. These molecules are recognized by surface receptors that are referred to as pattern recognition receptors. Oligogalacturonides (OGs), DAMPs released from the plant cell wall homogalacturonan, have also been proposed to act as local signals in the response to wounding. The Arabidopsis Wall-Associated Kinase 1 (WAK1), a receptor of OGs, has been described to form a complex with a cytoplasmic plasma membrane-localized kinase-associated protein phosphatase (KAPP) and a glycine-rich protein (GRP-3) that we find localized mainly in the cell wall and, in a small part, on the plasma membrane. By using Arabidopsis plants overexpressing WAK1, and both grp-3 and kapp null insertional mutant and overexpressing plants, we demonstrate a positive function of WAK1 and a negative function of GRP-3 and KAPP in the OG-triggered expression of defence genes and the production of an oxidative burst. The three proteins also affect the local response to wounding and the basal resistance against the necrotrophic pathogen Botrytis cinerea. GRP-3 and KAPP are likely to function in the phasing out of the plant immune response.

Expression analysis of self-incompatibility-associated genes in non-heading Chinese cabbage.

In Brassicaceae, a self-incompatibility (SI) system mediates pollen-pistil interactions. Self-pollen could be recognized and rejected by incompatible pistils. Several components involved in the SI response have been determined, including S-locus receptor kinase (SRK), S-locus cysteine-rich protein/S-locus protein 11, and arm repeat-containing protein 1 (ARC1). However, the components involved in the SI system of Brassicaceae are not fully understood. Here, we detected expression patterns of 24 SI-related genes in non-heading Chinese cabbage (Brassica campestris ssp chinensis Makino) after compatible and incompatible pollination, and potential interaction relationships of these genes were predicted. SRK and ARC1 transcripts increased initially 0.25 h after incompatible pollination, while kinase-associated protein phosphatase had an expression pattern that was opposite that of SRK transcripts during self-pollination. Plant U-box 8 was not required in the SI response of non-heading Chinese cabbage. Our results showed that the SI signal of non-heading Chinese cabbage could occur within 0.25 h after self-pollination. The hypothetical interaction relationships indicated that plastid-lipid-associated protein and malate dehydrogenase could be negatively regulated by chaperonin 10, glutathione transferase, cytidylate kinase/uridylate kinase, and methionine synthase by indirect interactions. Our findings could be helpful to better understand potential roles of these components in the SI system of non-heading Chinese cabbage.

Cyclin-dependent kinase-associated protein phosphatase is overexpressed in alcohol-related hepatocellular carcinoma and influences xenograft tumor growth

The cyclin-dependent kinase (Cdk)-associated protein phosphatase (KAP) is a dual-specificity phosphatase that dephosphorylates Cdk2 and inhibits cell cycle progression. The overexpression of KAP has been found in breast, prostate and renal cell carcinomas. However, the role of KAP in hepatocellular carcinoma (HCC) remains unclear. Therefore, the aim of this study was to investigate the expression of KAP in HCC and elucidate its role in tumorigenesis. HCC tissues from 117 patients undergoing surgical resection were collected for western blot analysis and immunohistochemichal analysis to establish clinical correlation. The antisense-mediated inhibition of KAP expression was performed in Huh-7 cell lines for tumorigenicity and growth regulation experiments. Clinicopathological analysis indicated that KAP was overexpressed in HCC tissue from alcoholic patients (P<0.001). It was significantly overexpressed in patients with a tumor number of <3 (P=0.0271), suggesting the potential role of KAP in tumorigenesis during early-stage alcohol-related HCC. Additionally, the antisense-mediated inhibition of KAP in Huh-7 HCC cells interfered with cell cycle progression, decreased cell proliferation, reduced the colony-forming ability of the cells and increased apoptosis. Tumorigenicity experiments showed that the KAP knockdown in Huh-7 cells generated smaller tumors in nude mice compared with the mock controls (P=0.018). In the cells in which KAP had been knocked down, the physical interaction between KAP and Cdk2 significantly increased, despite the reduced expression levels of KAP. The phosphorylation of cell proliferation and apoptosis-associated proteins, including phosphatase and tensin homolog (PTEN), glycogen synthase kinase (GSK), p44/42 and Akt, was decreased. Therefore, it can be concluded that KAP is overexpressed in alcohol-related HCC. The antisense-mediated knockdown of KAP in Huh-7 cells decreased cell proliferation, reduced the colony-forming ability of the cells, interfered with cell cycle progression and suppressed xenograft tumor formation, partly through enhanced KAP and Cdk2 interaction.

Overexpression of cyclin-dependent kinase-associated protein phosphatase enhances cell proliferation in renal cancer cells

ObjectiveThe aim of this study was to understand the role of cyclin-dependent kinase-associated protein phosphatase (KAP) in renal cancer cell growth. Materials and methodsRenal cell carcinoma (RCC) tissues from 58 patients receiving surgical resection were included for immunohistochemistry analysis. Additionally, human embryonic kidney (HEK293) cells overexpressing KAP were established for tumorigenicity experiments. ResultsClinicopathologic analysis indicated that poorly differentiated RCCs with a higher histological grade (grade 3/4) were associated with a higher proportion of KAP-positive cells (P < 0.001) as well as cytoplasmic expression of KAP (P < 0.05). HEK293 cells overexpressing KAP had a higher growth rate, greater resistance to TNF-α mediated increment of caspase 3 activity, a shorter cell cycle time, and greater ability of cell invasion. Tumorigenicity experiments showed that KAP-overexpressing cells generated significantly larger xenograft tumors in nude mice compared with mock controls (P = 0.032). ConclusionsKAP expression was associated with poorly differentiated RCCs and overexpression of KAP in renal cells enhanced cell proliferation, resistance to apoptosis, invasive ability, and xenograft tumor formation.

Identification of Target Ligands of CORYNE in Arabidopsis by Phage Display Library

CORYNE (CRN) plays important roles in stem cell division and differentiation of shoot apical meristem (SAM) in Arabidopsis thaliana. The cytoplasmic kinase domain of CRN has been cloned and expressed in Escherichia coli, and further purified by two consecutive steps of affinity chromatography. By using this purified CRN as a ligand, a 12-mer random-peptide library was used to determine the specific amino acid sequences binding with the recombinant CRN molecule. After four rounds of biopanning, positive phage clones were isolated and sequenced, and further tested by enzyme linked immunosorbent assay for their binding ability and specificity. Two positive clones that specifically bind to the intracellular protein kinase domain of CRN have been identified. Alignment of these peptides and the kinase-associated protein phosphatase (KAPP) shows high similarity, indicating that KAPP might interact with the cytoplasmic kinase domain of CRN and negatively regulate the CLV signal. Our current study would be helpful to better understand the CLV3 signal pathway.

Plasma Membrane Receptor Complexes

Recent data on the plasma membrane (PM)-located LRR-RLKs (for Leu-rich repeat receptor-like kinases) BRI1 (for brassinosteroid insensitive 1) and the coreceptors BAK1 (for BRI1-associated kinase 1) and SERK1 (for somatic embryogenesis receptor-like kinase 1) that participate in the perception of brassinosteroids (BRs) suggest that they are organized into heterooligomeric protein complexes. Other components of this complex include members of the 14-3-3 family, and, in the case of SERK1, the kinase-associated protein phosphatase (KAPP) and the AAA ATPase cell division cycle 48A (CDC48A). CDC48 proteins interact with ubiquitinated target proteins in animal and plant cells. In this Update we describe the role of several of the nonreceptor partners of the PM receptor complex with an emphasis on the role of CDC48 proteins in translocation and ubiquitination as a proposed mode of regulation of plant PM receptors.

The Arabidopsis Kinase-Associated Protein Phosphatase Regulates Adaptation to Na+ Stress

The kinase-associated protein phosphatase (KAPP) is a regulator of the receptor-like kinase (RLK) signaling pathway. Loss-of-function mutations rag1-1 (root attenuated growth1-1) and rag1-2, in the locus encoding KAPP, cause NaCl hypersensitivity in Arabidopsis thaliana. The NaCl hypersensitive phenotype exhibited by rag1 seedlings includes reduced shoot and primary root growth, root tip swelling, and increased lateral root formation. The phenotype exhibited by rag1-1 seedlings is associated with a specific response to Na(+) toxicity. The sensitivity to Na(+) is Ca(2+) independent and is not due to altered intracellular K(+)/Na(+). Analysis of the genetic interaction between rag1-1 and salt overly sensitive1 (sos1-14) revealed that KAPP is not a component of the SOS signal transduction pathway, the only Na(+) homeostasis signaling pathway identified so far in plants. All together, these results implicate KAPP as a functional component of the RLK signaling pathway, which also mediates adaptation to Na(+) stress. RLK pathway components, known to be modulated by NaCl at the messenger RNA level, are constitutively down-regulated in rag1-1 mutant plants. The effect of NaCl on their expression is not altered by the rag1-1 mutation.

Phosphoprotein and Phosphopeptide Interactions with the FHA Domain from Arabidopsis Kinase-Associated Protein Phosphatase

FHA domains are phosphoThr recognition modules found in diverse signaling proteins, including kinase-associated protein phosphatase (KAPP) from Arabidopsis thaliana. The kinase-interacting FHA domain (KI-FHA) of KAPP targets it to function as a negative regulator of some receptor-like kinase (RLK) signaling pathways important in plant development and environmental responses. To aid in the identification of potential binding sites for the KI-FHA domain, we predicted (i) the structure of a representative KAPP-binding RLK, CLAVATA1, and (ii) the functional surfaces of RLK kinase domains using evolutionary trace analysis. We selected phosphopeptides from KAPP-binding Arabidopsis RLKs for in vitro studies of association with KI-FHA from KAPP. Three phosphoThr peptide fragments from the kinase domain of CLV1 or BAK1 were found to bind KI-FHA with KD values of 8-20 microM, by NMR or titration calorimetry. Their affinity is driven by favorable enthalpy and solvation entropy gain. Mutagenesis of these three threonine sites suggests Thr546 in the C-lobe of the BAK1 kinase domain to be a principal but not sole site of KI-FHA binding in vitro. The brassinosteroid receptor BRI1 and KAPP are shown to associate in vivo and in vitro. Further genetic studies indicate that KAPP may be a negative regulator of the BRI1 signaling transduction pathway. 15N-Labeled KI-FHA was titrated with the GST-BRI1 kinase domain and monitored by NMR. BRI1 interacts with the same 3/4, 4/5, 6/7, 8/9, and 10/11 recognition loops of KI-FHA, with similar affinity as the phosphoThr peptides.

Partially Unfolded Forms and Non-two-state Folding of a β-Sandwich: FHA Domain from Arabidopsis Receptor Kinase-associated Protein Phosphatase

FHA domains adopt a β-sandwich fold with 11 strands. The first evidence of partially unfolded forms of a β-sandwich is derived from native-state hydrogen exchange (NHX) of the forkhead-associated (FHA) domain from kinase-associated protein phosphatase from Arabidopsis. The folding kinetics of this FHA domain indicate that EX2 behavior prevails at pH 6.3. In the chevron plot, rollover in the folding arm and bends in the unfolding arm suggest folding intermediates. NHX of this FHA domain suggests a core of six most stable β-strands and two loops, characterized by rare global unfolding events. Flanking this stable core are β-strands and recognition loops with less stability, termed subglobal motifs. These suggest partially unfolded forms (near-native intermediates) with two levels of stability. The spatial separation of the subglobal motifs on the flanks suggests possible parallelism in their folding as additional β-strands align with the stable core of six strands. Intermediates may contribute to differences in stabilities and m-values suggested by NHX or kinetics relative to chemical denaturation. Residual structure in the unfolded regime is suggested by superprotection of β-strand 6 and by GdmCl-dependence of adjustments in amide NMR spectra and residual optical signal. The global folding stability depends strongly on pH, with at least 3 kcal/mol more stability at pH 7.3 than at pH 6.3. This FHA domain is hypothesized to fold progressively with initial hydrophobic collapse of its stable six-stranded core followed by addition of less stable flanking β-strands and ordering of recognition loops.

DAWDLE, a Forkhead-Associated Domain Gene, Regulates Multiple Aspects of Plant Development

Phosphoprotein-binding domains are found in many different proteins and specify protein-protein interactions critical for signal transduction pathways. Forkhead-associated (FHA) domains bind phosphothreonine and control many aspects of cell proliferation in yeast (Saccharomyces cerevisiae) and animal cells. The Arabidopsis (Arabidopsis thaliana) protein kinase-associated protein phosphatase includes a FHA domain that mediates interactions with receptor-like kinases, which in turn regulate a variety of signaling pathways involved in plant growth and pathogen responses. Screens for insertional mutations in other Arabidopsis FHA domain-containing genes identified a mutant with pleiotropic defects. dawdle (ddl) plants are developmentally delayed, produce defective roots, shoots, and flowers, and have reduced seed set. DDL is expressed in the root and shoot meristems and the reduced size of the root apical meristem in ddl plants suggests a role early in organ development.

FHA Domain of Kinase Associated Protein Phosphatase (KAPP): Interactions with PhosphoThr Peptides and a Receptor Kinase and Dynamics by NMR

FHA Domain of Kinase Associated Protein Phosphatase (KAPP): Interactions with PhosphoThr Peptides and a Receptor Kinase and Dynamics by NMR

TheArabidopsisSOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE1 Protein Complex Includes BRASSINOSTEROID-INSENSITIVE1

Arabidopsis thaliana SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE1 (SERK1) is a leucine-rich repeat receptor-like kinase (LRR-RLK) involved in the acquisition of embryogenic competence and in male sporogenesis. To determine the composition of the SERK1 signaling complex in vivo, we generated plants expressing the SERK1 protein fused to cyan fluorescent protein under SERK1 promoter control. The membrane receptor complex was immunoprecipitated from seedlings, and the coimmunoprecipitating proteins were identified using liquid chromatography/matrix-assisted laser desorption ionization-time of flight/mass spectrometry of the trypsin-released peptides. This approach identified two other LRR-RLKs, the BRASSINOSTEROID-INSENSITIVE1 (BRI1) receptor and its coreceptor, the SERK3 or BRI1-ASSOCIATED KINASE1 protein. In addition, KINASE-ASSOCIATED PROTEIN PHOSPHATASE, CDC48A, and 14-3-3nu were found. Finally, the MADS box transcription factor AGAMOUS-LIKE15 and an uncharacterized zinc finger protein, a member of the CONSTANS family, were identified as part of the SERK1 complex. Using blue native gel electrophoresis, we show that SERK1 and SERK3 are part of BRI1-containing multiple protein complexes with relative masses between 300 and 500 kD. The SERK1 mutant allele serk1-1 enhances the phenotype of the weak BRI1 allele bri1-119. Collectively, these results suggest that apart from SERK3, SERK1 is also involved in the brassinolide signaling pathway.

PhosphoThr Peptide Binding Globally Rigidifies Much of the FHA Domain from Arabidopsis Receptor Kinase-Associated Protein Phosphatase,

A net increase in the backbone rigidity of the kinase-interacting FHA domain (KI-FHA) from the Arabidopsis receptor kinase-associated protein phosphatase (KAPP) accompanies the binding of a phosphoThr peptide from its CLV1 receptor-like kinase partner, according to (15)N NMR relaxation at 11.7 and 14.1 T. All of the loops of free KI-FHA display evidence of nanosecond-scale motions. Many of these same residues have residual dipolar couplings that deviate from structural predictions. Binding of the CLV1 pT868 peptide seems to reduce nanosecond-scale fluctuations of all loops, including half of the residues of recognition loops. Residues important for affinity are found to be rigid, i.e., conserved residues and residues of the subsite for the key pT+3 peptide position. This behavior parallels SH2 and PTB domain recognition of pTyr peptides. PhosphoThr peptide binding increases KI-FHA backbone rigidity (S(2)) of three recognition loops, a loop nearby, seven strands from the beta-sandwich, and a distal loop. Compensating the trend of increased rigidity, binding enhances fast mobility at a few sites in four loops on the periphery of the recognition surface and in two loops on the far side of the beta-sandwich. Line broadening evidence of microsecond- to millisecond-scale fluctuations occurs across the six-stranded beta-sheet and nearby edges of the beta-sandwich; this forms a network connected by packing of interior side chains and H-bonding. A patch of the slowly fluctuating residues coincides with the site of segment-swapped dimerization in crystals of the FHA domain of human Chfr. Phosphopeptide binding introduces microsecond- to millisecond-scale fluctuations to more residues of the long 8/9 recognition loop of KI-FHA. The rigidity of this FHA domain appears to couple as a whole to pThr peptide binding.

Interaction of calmodulin, a sorting nexin and kinase-associated protein phosphatase with the Brassica oleracea S locus receptor kinase.

Recognition of self-pollen during the self-incompatibility response in Brassica oleracea is mediated by the binding of a secreted peptide (the S locus cysteine-rich protein) to the S locus receptor kinase (SRK), a member of the plant receptor kinase (PRK) superfamily. Here, we describe the characterization of three proteins that interact with the cytosolic kinase domain of SRK. A B. oleracea homolog of Arabidopsis kinase-associated protein phosphatase was shown to interact with and dephosphorylate SRK and was itself phosphorylated by SRK. Yeast (Saccharomyces cerevisiae) two-hybrid screens identified two additional interactors, calmodulin and a sorting nexin, both of which have been implicated in receptor kinase down-regulation in animals. A calmodulin-binding site was identified in sub-domain VIa of the SRK kinase domain. The binding site is conserved and functional in several other members of the PRK family. The sorting nexin also interacted with diverse members of the PRK family, suggesting that all three of the interacting proteins described here may play a general role in signal transduction by this family of proteins.

NMR structure of the forkhead-associated domain from the Arabidopsis receptor kinase-associated protein phosphatase.

Forkhead-associated (FHA) domains are phosphoprotein-binding modules found in diverse signaling proteins that bind partners phosphorylated on threonine or serine. Kinase-associated protein phosphatase from Arabidopsis employs its FHA domain for negative regulation of receptor-like kinase signaling pathways, which are important in plant development. The solution structure of the free state of kinase-interacting FHA domain (KI-FHA) of kinase-associated protein phosphatase has been determined with high precision and accuracy using residual dipolar couplings. KI-FHA is a sandwich of a five-stranded mixed beta-sheet with a six-stranded antiparallel beta-sheet. Despite homology only in the recognition loops, this fold is shared with FHA domains from checkpoint proteins from yeast and humans, as well as with nonhomologous MH2 domains of Smad tumor suppressors. A shared pattern of hydrophobicity throughout FHA domains and Smad MH2 domains may stabilize the core of the beta-sandwich. Evolutionary trace analysis of FHA domains suggests class-specific residues in the recognition loops that could tune their phosphoprotein-binding specificity. This surface agrees with that of KI-FHA in contact with a phosphothreonine peptide ligand. Evolutionary trace analysis also predicts an unexpected swath of class-specific residues on another face of FHA domains. Protein interactions with these faces may affect assembly of transmembrane signaling complexes in plants, and in other FHA domain-containing assemblies.

The Arabidopsis kinase-associated protein phosphatase controls internalization of the somatic embryogenesis receptor kinase 1.

The AtSERK1 protein is a plasma membrane-located LRR receptor-like serine threonine kinase that is transiently expressed during plant embryogenesis. Our results show that AtSERK1 interacts with the kinase-associated protein phosphatase (KAPP) in vitro. The kinase interaction (KI) domain of KAPP does not interact with a catalytically inactive kinase mutant. Using mutant AtSERK1 proteins in which Thr 462, Thr 463, and Thr 468 in the A-loop of the AtSERK1 kinase domain were replaced by alanines, we show that phosphorylation status of the receptor is involved in interaction with KAPP. KAPP and AtSERK1 cDNAs were fused to two different variants of green fluorescent protein (GFP), the yellow fluorescent protein (YFP) or the cyan fluorescent protein (CFP). Both KAPP and AtSERK1 proteins are found at the plasma membrane. Our results show that AtSERK1-CFP becomes sequestered into intracellular vesicles when transiently coexpressed with KAPP-YFP proteins. AtSERK1(T463A)-CFP and AtSERK1(3T-->A)-CFP proteins were partially sequestered intracellularly in the absence of KAPP-YFP protein, suggesting an active role for KAPP dephosphorylation of threonine residues in the AtSERK1 A-loop in receptor internalization. The interaction between the KAPP-CFP/YFP and AtSERK1-CFP/YFP fusion proteins was investigated with fluorescence spectral imaging microscopy (FSPIM). Our results show that AtSERK1-CFP and KAPP-YFP proteins are colocalized at the plasma membrane but only show fluorescence energy transfer (FRET) indicative of physical interaction in intracellular vesicles. These results suggest that KAPP is an integral part of the AtSERK1 endocytosis mechanism.

Interaction of the Arabidopsis Receptor Protein Kinase Wak1 with a Glycine-rich Protein, AtGRP-3

The Arabidopsis wall-associated receptor kinase, Wak1, is a member of the Wak family (Wak1-5) that links the plasma membrane to the extracellular matrix. By the yeast two-hybrid screen, we found that a glycine-rich extracellular protein, AtGRP-3, binds to the extracellular domain of Wak1. Further in vitro binding studies indicated that AtGRP-3 is the only isoform among the six tested AtGRPs that specifically interacts with Waks, and the cysteine-rich carboxyl terminus of AtGRP-3 is essential for its binding to Wak1. We also show that Wak1 and AtGRP-3 form a complex with a molecular size of approximately 500 kDa in vivo in conjunction with the kinase-associated protein phosphatase, KAPP, that has been shown to interact with a number of plant receptor-like kinases. Binding of AtGRP-3 to Wak1 is shown to be crucial for the integrity of the complex. Wak1 and AtGRP-3 are both induced by salicylic acid treatment. Moreover, exogenously added AtGRP-3 up-regulates the expression of Wak1, AtGRP-3, and PR-1 (for pathogenesis-related) in protoplasts. Taken together, our data suggest that AtGRP-3 regulates Wak1 function through binding to the cell wall domain of Wak1 and that the interaction of Wak1 with AtGRP-3 occurs in a pathogenesis-related process in planta.