Decoding Arabidopsis thaliana CPK/SnRK Superfamily Kinase Client Signaling Networks Using Peptide Library and Mass Spectrometry.

Members of the calcium-dependent protein kinase (CDPK/CPK) and SNF-related protein kinase (SnRK) superfamilies are commonly found in plants and some protists. Our knowledge of client specificity of the members of this superfamily is fragmentary. As this family is represented by over 30 members in Arabidopsis thaliana, the identification of kinase-specific and overlapping client relationships is crucial to our understanding the nuances of this large family of kinases as directed towards signal transduction pathways. Herein, we used the kinase client (KiC) assay-a relative, quantitative, high-throughput mass spectrometry-based in vitro phosphorylation assay-to identify and characterize potential CPK/SnRK targets of Arabidopsis. Eight CPKs (1, 3, 6, 8, 17, 24, 28, and 32), four SnRKs (subclass 1 and 2), and PPCK1 and PPCK2 were screened against a synthetic peptide library that contains 2095 peptides and 2661 known phosphorylation sites. A total of 625 in vitro phosphorylation sites corresponding to 203 non-redundant proteins were identified. The most promiscuous kinase, CPK17, had 105 candidate target proteins, many of which had already been discovered. Sequence analysis of the identified phosphopeptides revealed four motifs: LxRxxS, RxxSxxR, RxxS, and LxxxxS, that were significantly enriched among CPK/SnRK clients. The results provide insight into both CPK- and SnRK-specific and overlapping signaling network architectures and recapitulate many known in vivo relationships validating this large-scale approach towards discovering kinase targets.

Insights into the ISG15 transfer cascade by the UBE1L activating enzyme.

The attachment of the ubiquitin-like protein ISG15 to substrates by specific E1-E2-E3 enzymes is a well-established signalling mechanism of the innate immune response. Here, we present a 3.45 Å cryo-EM structure of a chemically trapped UBE1L-UBE2L6 complex bound to activated ISG15. This structure reveals the details of the first steps of ISG15 recognition and UBE2L6 recruitment by UBE1L (also known as UBA7). Taking advantage of viral effector proteins from severe acute respiratory coronavirus 2 (SARS-CoV-2) and influenza B virus (IBV), we validate the structure and confirm the importance of the ISG15 C-terminal ubiquitin-like domain in the adenylation reaction. Moreover, biochemical characterization of the UBE1L-ISG15 and UBE1L-UBE2L6 interactions enables the design of ISG15 and UBE2L6 mutants with altered selectively for the ISG15 and ubiquitin conjugation pathways. Together, our study helps to define the molecular basis of these interactions and the specificity determinants that ensure the fidelity of ISG15 signalling during the antiviral response.

ISGylation-independent protection of cell growth by USP18 following interferon stimulation.

Type 1 interferon stimulation highly up-regulates all elements of a ubiquitin-like conjugation system that leads to ISGylation of target proteins. An ISG15-specific member of the deubiquitylase family, USP18, is up-regulated in a co-ordinated manner. USP18 can also provide a negative feedback by inhibiting JAK-STAT signalling through protein interactions independently of DUB activity. Here, we provide an acute example of this phenomenon, whereby the early expression of USP18, post-interferon treatment of HCT116 colon cancer cells is sufficient to fully suppress the expression of the ISG15 E1 enzyme, UBA7. Stimulation of lung adenocarcinoma A549 cells with interferon reduces their growth rate but they remain viable. In contrast, A549 USP18 knock-out cells show similar growth characteristics under basal conditions, but upon interferon stimulation, a profound inhibition of cell growth is observed. We show that this contingency on USP18 is independent of ISGylation, suggesting non-catalytic functions are required for viability. We also demonstrate that global deISGylation kinetics are very slow compared with deubiquitylation. This is not influenced by USP18 expression, suggesting that enhanced ISGylation in USP18 KO cells reflects increased conjugating activity.

Dissecting distinct proteolytic activities of FMDV Lpro implicates cleavage and degradation of RLR signaling proteins, not its deISGylase/DUB activity, in type I interferon suppression.

The type I interferon response is an important innate antiviral pathway. Recognition of viral RNA by RIG-I-like receptors (RLRs) activates a signaling cascade that leads to type I interferon (IFN-α/ß) gene transcription. Multiple proteins in this signaling pathway (e.g. RIG-I, MDA5, MAVS, TBK1, IRF3) are regulated by (de)ubiquitination events. Most viruses have evolved mechanisms to counter this antiviral response. The leader protease (Lpro) of foot-and-mouth-disease virus (FMDV) has been recognized to reduce IFN-α/ß gene transcription; however, the exact mechanism is unknown. The proteolytic activity of Lpro is vital for releasing itself from the viral polyprotein and for cleaving and degrading specific host cell proteins, such as eIF4G and NF-κB. In addition, Lpro has been demonstrated to have deubiquitination/deISGylation activity. Lpro's deubiquitination/deISGylation activity and the cleavage/degradation of signaling proteins have both been postulated to be important for reduced IFN-α/ß gene transcription. Here, we demonstrate that TBK1, the kinase that phosphorylates and activates the transcription factor IRF3, is cleaved by Lpro in FMDV-infected cells as well as in cells infected with a recombinant EMCV expressing Lpro. In vitro cleavage experiments revealed that Lpro cleaves TBK1 at residues 692-694. We also observed cleavage of MAVS in HeLa cells infected with EMCV-Lpro, but only observed decreasing levels of MAVS in FMDV-infected porcine LFPK αVß6 cells. We set out to dissect Lpro's ability to cleave RLR signaling proteins from its deubiquitination/deISGylation activity to determine their relative contributions to the reduction of IFN-α/ß gene transcription. The introduction of specific mutations, of which several were based on the recently published structure of Lpro in complex with ISG15, allowed us to identify specific amino acid substitutions that separate the different proteolytic activities of Lpro. Characterization of the effects of these mutations revealed that Lpro's ability to cleave RLR signaling proteins but not its deubiquitination/deISGylation activity correlates with the reduced IFN-ß gene transcription.

The Tumour Suppressor TMEM127 Is a Nedd4-Family E3 Ligase Adaptor Required by Salmonella SteD to Ubiquitinate and Degrade MHC Class II Molecules.

The Salmonella enterica effector SteD depletes mature MHC class II (mMHCII) molecules from the surface of infected antigen-presenting cells through ubiquitination of the cytoplasmic tail of the mMHCII ß chain. Here, through a genome-wide mutant screen of human antigen-presenting cells, we show that the NEDD4 family HECT E3 ubiquitin ligase WWP2 and a tumor-suppressing transmembrane protein of unknown biochemical function, TMEM127, are required for SteD-dependent ubiquitination of mMHCII. Although evidently not involved in normal regulation of mMHCII, TMEM127 was essential for SteD to suppress both mMHCII antigen presentation in mouse dendritic cells and MHCII-dependent CD4+ T cell activation. We found that TMEM127 contains a canonical PPxY motif, which was required for binding to WWP2. SteD bound to TMEM127 and enabled TMEM127 to interact with and induce ubiquitination of mature MHCII. Furthermore, SteD also underwent TMEM127- and WWP2-dependent ubiquitination, which both contributed to its degradation and augmented its activity on mMHCII.

Global Landscape and Dynamics of Parkin and USP30-Dependent Ubiquitylomes in iNeurons during Mitophagic Signaling.

The ubiquitin ligase Parkin, protein kinase PINK1, USP30 deubiquitylase, and p97 segregase function together to regulate turnover of damaged mitochondria via mitophagy, but our mechanistic understanding in neurons is limited. Here, we combine induced neurons (iNeurons) derived from embryonic stem cells with quantitative proteomics to reveal the dynamics and specificity of Parkin-dependent ubiquitylation under endogenous expression conditions. Targets showing elevated ubiquitylation in USP30-/- iNeurons are concentrated in components of the mitochondrial translocon, and the ubiquitylation kinetics of the vast majority of Parkin targets are unaffected, correlating with a modest kinetic acceleration in accumulation of pS65-Ub and mitophagic flux upon mitochondrial depolarization without USP30. Basally, ubiquitylated translocon import substrates accumulate, suggesting a quality control function for USP30. p97 was dispensable for Parkin ligase activity in iNeurons. This work provides an unprecedented quantitative landscape of the Parkin-modified ubiquitylome in iNeurons and reveals the underlying specificity of central regulatory elements in the pathway.

Insights into ubiquitin chain architecture using Ub-clipping.

Protein ubiquitination is a multi-functional post-translational modification that affects all cellular processes. Its versatility arises from architecturally complex polyubiquitin chains, in which individual ubiquitin moieties may be ubiquitinated on one or multiple residues, and/or modified by phosphorylation and acetylation1-3. Advances in mass spectrometry have enabled the mapping of individual ubiquitin modifications that generate the ubiquitin code; however, the architecture of polyubiquitin signals has remained largely inaccessible. Here we introduce Ub-clipping as a methodology by which to understand polyubiquitin signals and architectures. Ub-clipping uses an engineered viral protease, Lbpro∗, to incompletely remove ubiquitin from substrates and leave the signature C-terminal GlyGly dipeptide attached to the modified residue; this simplifies the direct assessment of protein ubiquitination on substrates and within polyubiquitin. Monoubiquitin generated by Lbpro∗ retains GlyGly-modified residues, enabling the quantification of multiply GlyGly-modified branch-point ubiquitin. Notably, we find that a large amount (10-20%) of ubiquitin in polymers seems to exist as branched chains. Moreover, Ub-clipping enables the assessment of co-existing ubiquitin modifications. The analysis of depolarized mitochondria reveals that PINK1/parkin-mediated mitophagy predominantly exploits mono- and short-chain polyubiquitin, in which phosphorylated ubiquitin moieties are not further modified. Ub-clipping can therefore provide insight into the combinatorial complexity and architecture of the ubiquitin code.

OTULIN deficiency in ORAS causes cell type-specific LUBAC degradation, dysregulated TNF signalling and cell death.

The deubiquitinase OTULIN removes methionine-1 (M1)-linked polyubiquitin signals conjugated by the linear ubiquitin chain assembly complex (LUBAC) and is critical for preventing TNF-driven inflammation in OTULIN-related autoinflammatory syndrome (ORAS). Five ORAS patients have been reported, but how dysregulated M1-linked polyubiquitin signalling causes their symptoms is unclear. Here, we report a new case of ORAS in which an OTULIN-Gly281Arg mutation leads to reduced activity and stability in vitro and in cells. In contrast to OTULIN-deficient monocytes, in which TNF signalling and NF-κB activation are increased, loss of OTULIN in patient-derived fibroblasts leads to a reduction in LUBAC levels and an impaired response to TNF Interestingly, both patient-derived fibroblasts and OTULIN-deficient monocytes are sensitised to certain types of TNF-induced death, and apoptotic cells are evident in ORAS patient skin lesions. Remarkably, haematopoietic stem cell transplantation leads to complete resolution of inflammatory symptoms, including fevers, panniculitis and diarrhoea. Therefore, haematopoietic cells are necessary for clinical manifestation of ORAS Together, our data suggest that ORAS pathogenesis involves hyper-inflammatory immune cells and TNF-induced death of both leukocytes and non-haematopoietic cells.

A Chlamydia effector combining deubiquitination and acetylation activities induces Golgi fragmentation.

Pathogenic bacteria are armed with potent effector proteins that subvert host signalling processes during infection1. The activities of bacterial effectors and their associated roles within the host cell are often poorly understood, particularly for Chlamydia trachomatis2, a World Health Organization designated neglected disease pathogen. We identify and explain remarkable dual Lys63-deubiquitinase (DUB) and Lys-acetyltransferase activities in the Chlamydia effector ChlaDUB1. Crystal structures capturing intermediate stages of each reaction reveal how the same catalytic centre of ChlaDUB1 can facilitate such distinct processes, and enable the generation of mutations that uncouple the two activities. Targeted Chlamydia mutant strains allow us to link the DUB activity of ChlaDUB1 and the related, dedicated DUB ChlaDUB2 to fragmentation of the host Golgi apparatus, a key process in Chlamydia infection for which effectors have remained elusive. Our work illustrates the incredible versatility of bacterial effector proteins, and provides important insights towards understanding Chlamydia pathogenesis.

Irreversible inactivation of ISG15 by a viral leader protease enables alternative infection detection strategies.

In response to viral infection, cells mount a potent inflammatory response that relies on ISG15 and ubiquitin posttranslational modifications. Many viruses use deubiquitinases and deISGylases that reverse these modifications and antagonize host signaling processes. We here reveal that the leader protease, Lbpro, from foot-and-mouth disease virus (FMDV) targets ISG15 and to a lesser extent, ubiquitin in an unprecedented manner. Unlike canonical deISGylases that hydrolyze the isopeptide linkage after the C-terminal GlyGly motif, Lbpro cleaves the peptide bond preceding the GlyGly motif. Consequently, the GlyGly dipeptide remains attached to the substrate Lys, and cleaved ISG15 is rendered incompetent for reconjugation. A crystal structure of Lbpro bound to an engineered ISG15 suicide probe revealed the molecular basis for ISG15 proteolysis. Importantly, anti-GlyGly antibodies, developed for ubiquitin proteomics, are able to detect Lbpro cleavage products during viral infection. This opens avenues for infection detection of FMDV based on an immutable, host-derived epitope.

Ubiquitin-Mediated Regulation of RIPK1 Kinase Activity Independent of IKK and MK2.

Tumor necrosis factor (TNF) can drive inflammation, cell survival, and death. While ubiquitylation-, phosphorylation-, and nuclear factor κB (NF-κB)-dependent checkpoints suppress the cytotoxic potential of TNF, it remains unclear whether ubiquitylation can directly repress TNF-induced death. Here, we show that ubiquitylation regulates RIPK1's cytotoxic potential not only via activation of downstream kinases and NF-kB transcriptional responses, but also by directly repressing RIPK1 kinase activity via ubiquitin-dependent inactivation. We find that the ubiquitin-associated (UBA) domain of cellular inhibitor of apoptosis (cIAP)1 is required for optimal ubiquitin-lysine occupancy and K48 ubiquitylation of RIPK1. Independently of IKK and MK2, cIAP1-mediated and UBA-assisted ubiquitylation suppresses RIPK1 kinase auto-activation and, in addition, marks it for proteasomal degradation. In the absence of a functional UBA domain of cIAP1, more active RIPK1 kinase accumulates in response to TNF, causing RIPK1 kinase-mediated cell death and systemic inflammatory response syndrome. These results reveal a direct role for cIAP-mediated ubiquitylation in controlling RIPK1 kinase activity and preventing TNF-mediated cytotoxicity.

Ubiquitin Linkage-Specific Affimers Reveal Insights into K6-Linked Ubiquitin Signaling.

Several ubiquitin chain types have remained unstudied, mainly because tools and techniques to detect these posttranslational modifications are scarce. Linkage-specific antibodies have shaped our understanding of the roles and dynamics of polyubiquitin signals but are available for only five out of eight linkage types. We here characterize K6- and K33-linkage-specific "affimer" reagents as high-affinity ubiquitin interactors. Crystal structures of affimers bound to their cognate chain types reveal mechanisms of specificity and a K11 cross-reactivity in the K33 affimer. Structure-guided improvements yield superior affinity reagents suitable for western blotting, confocal fluorescence microscopy and pull-down applications. This allowed us to identify RNF144A and RNF144B as E3 ligases that assemble K6-, K11-, and K48-linked polyubiquitin in vitro. A protocol to enrich K6-ubiquitinated proteins from cells identifies HUWE1 as a main E3 ligase for this chain type, and we show that mitofusin-2 is modified with K6-linked polyubiquitin in a HUWE1-dependent manner.

Regulation of the Regulators: Post-Translational Modifications, Subcellular, and Spatiotemporal Distribution of Plant 14-3-3 Proteins.

14-3-3 proteins bind to and modulate the activity of phosphorylated proteins that regulate a variety of metabolic processes in eukaryotes. Multiple 14-3-3 isoforms are expressed in most organisms and display redundancy in both sequence and function. Plants contain the largest number of 14-3-3 isoforms. For example, Arabidopsis thaliana contains thirteen 14-3-3 genes, each of which is expressed. Interest in the plant 14-3-3 field has swelled over the past decade, largely due to the vast number of possibilities for 14-3-3 metabolic regulation. As the field progresses, it is essential to understand these proteins' activities at both the spatiotemporal and subcellular levels. This review summarizes current knowledge of 14-3-3 proteins in plants, including 14-3-3 interactions, regulatory functions, isoform specificity, and post-translational modifications. We begin with a historical overview and structural analysis of 14-3-3 proteins, which describes the basic principles of 14-3-3 function, and then discuss interactions and regulatory effects of plant 14-3-3 proteins in specific tissues and subcellular compartments. We conclude with a summary of 14-3-3 phosphorylation and current knowledge of the functional effects of this modification in plants.

Ubiquitin modifications.

Protein ubiquitination is a dynamic multifaceted post-translational modification involved in nearly all aspects of eukaryotic biology. Once attached to a substrate, the 76-amino acid protein ubiquitin is subjected to further modifications, creating a multitude of distinct signals with distinct cellular outcomes, referred to as the 'ubiquitin code'. Ubiquitin can be ubiquitinated on seven lysine (Lys) residues or on the N-terminus, leading to polyubiquitin chains that can encompass complex topologies. Alternatively or in addition, ubiquitin Lys residues can be modified by ubiquitin-like molecules (such as SUMO or NEDD8). Finally, ubiquitin can also be acetylated on Lys, or phosphorylated on Ser, Thr or Tyr residues, and each modification has the potential to dramatically alter the signaling outcome. While the number of distinctly modified ubiquitin species in cells is mind-boggling, much progress has been made to characterize the roles of distinct ubiquitin modifications, and many enzymes and receptors have been identified that create, recognize or remove these ubiquitin modifications. We here provide an overview of the various ubiquitin modifications present in cells, and highlight recent progress on ubiquitin chain biology. We then discuss the recent findings in the field of ubiquitin acetylation and phosphorylation, with a focus on Ser65-phosphorylation and its role in mitophagy and Parkin activation.

Assembly and specific recognition of k29- and k33-linked polyubiquitin.

Protein ubiquitination regulates many cellular processes via attachment of structurally and functionally distinct ubiquitin (Ub) chains. Several atypical chain types have remained poorly characterized because the enzymes mediating their assembly and receptors with specific binding properties have been elusive. We found that the human HECT E3 ligases UBE3C and AREL1 assemble K48/K29- and K11/K33-linked Ub chains, respectively, and can be used in combination with DUBs to generate K29- and K33-linked chains for biochemical and structural analyses. Solution studies indicate that both chains adopt open and dynamic conformations. We further show that the N-terminal Npl4-like zinc finger (NZF1) domain of the K29/K33-specific deubiquitinase TRABID specifically binds K29/K33-linked diUb, and a crystal structure of this complex explains TRABID specificity and suggests a model for chain binding by TRABID. Our work uncovers linkage-specific components in the Ub system for atypical K29- and K33-linked Ub chains, providing tools to further understand these unstudied posttranslational modifications.

Ubiquitin Ser65 phosphorylation affects ubiquitin structure, chain assembly and hydrolysis.

The protein kinase PINK1 was recently shown to phosphorylate ubiquitin (Ub) on Ser65, and phosphoUb activates the E3 ligase Parkin allosterically. Here, we show that PINK1 can phosphorylate every Ub in Ub chains. Moreover, Ser65 phosphorylation alters Ub structure, generating two conformations in solution. A crystal structure of the major conformation resembles Ub but has altered surface properties. NMR reveals a second phosphoUb conformation in which ß5-strand slippage retracts the C-terminal tail by two residues into the Ub core. We further show that phosphoUb has no effect on E1-mediated E2 charging but can affect discharging of E2 enzymes to form polyUb chains. Notably, UBE2R1- (CDC34), UBE2N/UBE2V1- (UBC13/UEV1A), TRAF6- and HOIP-mediated chain assembly is inhibited by phosphoUb. While Lys63-linked poly-phosphoUb is recognized by the TAB2 NZF Ub binding domain (UBD), 10 out of 12 deubiquitinases (DUBs), including USP8, USP15 and USP30, are impaired in hydrolyzing phosphoUb chains. Hence, Ub phosphorylation has repercussions for ubiquitination and deubiquitination cascades beyond Parkin activation and may provide an independent layer of regulation in the Ub system.

Multisite phosphorylation of 14-3-3 proteins by calcium-dependent protein kinases.

Plant 14-3-3 proteins are phosphorylated at multiple sites in vivo; however, the protein kinase(s) responsible are unknown. Of the 34 CPK (calcium-dependent protein kinase) paralogues in Arabidopsis thaliana, three (CPK1, CPK24 and CPK28) contain a canonical 14-3-3-binding motif. These three, in addition to CPK3, CPK6 and CPK8, were tested for activity against recombinant 14-3-3 proteins χ and ε. Using an MS-based quantitative assay we demonstrate phosphorylation of 14-3-3 χ and ε at a total of seven sites, one of which is an in vivo site discovered in Arabidopsis. CPK autophosphorylation was also comprehensively monitored by MS and revealed a total of 45 sites among the six CPKs analysed, most of which were located within the N-terminal variable and catalytic domains. Among these CPK autophosphorylation sites was Tyr463 within the calcium-binding EF-hand domain of CPK28. Of all CPKs assayed, CPK28, which contained an autophosphorylation site (Ser43) within a canonical 14-3-3-binding motif, showed the highest activity against 14-3-3 proteins. Phosphomimetic mutagenesis of Ser72 to aspartate on 14-3-3χ, which is adjacent to the 14-3-3-binding cleft and conserved among all 14-3-3 isoforms, prevented 14-3-3-mediated inhibition of phosphorylated nitrate reductase.

Initial description of the developing soybean seed protein Lys-N(ε)-acetylome.

Characterization of the myriad protein posttranslational modifications (PTM) is a key aspect of proteome profiling. While there have been previous studies of the developing soybean seed phospho-proteome, herein we present the first analysis of non-histone lysine-N(Ɛ)-acetylation in this system. In recent years there have been reports that lysine acetylation is widespread, affecting thousands of proteins in diverse species from bacteria to mammals. Recently preliminary descriptions of the protein lysine acetylome from the plants Arabidopsis thaliana and Vitis vinifera have been reported. Using a combination of immunoenrichment and mass spectrometry-based techniques, we have identified over 400 sites of lysine acetylation in 245 proteins from developing soybean (Glycine max (L.) Merr., cv. Jack) seeds, which substantially increases the number of known plant N(Ɛ)-lysine-acetylation sites. Results of functional annotation indicate acetyl-proteins are involved with a host of cellular activities. In addition to histones, and other proteins involved in RNA synthesis and processing, acetyl-proteins participate in signaling, protein folding, and a plethora of metabolic processes. Results from in silico localization indicate that lysine-acetylated proteins are present in all major subcellular compartments. In toto, our results establish developing soybean seeds as a physiologically distinct addendum to Arabidopsis thaliana seedlings for functional analysis of protein Lys-N(Ɛ)-acetylation. BIOLOGICAL SIGNIFICANCE: Several modes of peptide fragmentation and database search algorithms are incorporated to identify, for the first time, sites of lysine acetylation on a plethora of proteins from developing soybean seeds. The contributions of distinct techniques to achieve increased coverage of the lysine acetylome are compared, providing insight to their respective benefits. Acetyl-proteins and specific acetylation sites are characterized, revealing intriguing similarities as well as differences with those previously identified in other plant and non-plant species.

Purification of protein complexes and characterization of protein-protein interactions.

In plants and all other multicellular organisms, both the intra- and extracellular environments are filled with dynamic biomolecular interactions that control many biological processes. Most of these interactions are biochemical in nature and often exist between proteins. For instance, many protein-protein interactions assist in sustained cellular homeostasis but also allow for rapid intracellular communication in response to stimuli. Thus, the discovery and validation of protein-protein interactions, and the consequent formation of protein complexes, is an integral and essential component of plant biology research. The ability to efficiently and accurately determine existing protein networks is necessary to further our understanding of plant biology. However, discovering protein networks represents a challenge for both present and future researchers. Here we have outlined several straightforward methods aimed at first discovering protein-protein interactions and then characterizing them utilizing additional approaches. We first describe methods for rate-zonal centrifugation, in vitro binding assays, and co-immunoprecipitation experiments in the context of discovering novel protein-protein interactions. Next, we discuss methods for characterizing and validating these interactions using alternative approaches: yeast two-hybrid, in vitro pull-down assays, and bimolecular fluorescence complementation (BiFC). Obviously each of these methods need not be performed in parallel; rather our goal was to describe several approaches, some of which may be more appropriate for increasingly specialized laboratory environments.

A versatile mass spectrometry-based method to both identify kinase client-relationships and characterize signaling network topology.

While more than a thousand protein kinases (PK) have been identified in the Arabidopsis thaliana genome, relatively little progress has been made toward identifying their individual client proteins. Herein we describe the use of a mass spectrometry-based in vitro phosphorylation strategy, termed Kinase Client assay (KiC assay), to study a targeted-aspect of signaling. A synthetic peptide library comprising 377 in vivo phosphorylation sequences from developing seed was screened using 71 recombinant A. thaliana PK. Among the initial results, we identified 23 proteins as putative clients of 17 PK. In one instance protein phosphatase inhibitor-2 (AtPPI-2) was phosphorylated at multiple-sites by three distinct PK, casein kinase1-like 10, AME3, and a Ser PK-like protein. To confirm this result, full-length recombinant AtPPI-2 was reconstituted with each of these PK. The results confirmed multiple distinct phosphorylation sites within this protein. Biochemical analyses indicate that AtPPI-2 inhibits type 1 protein phosphatase (TOPP) activity, and that the phosphorylated forms of AtPPI-2 are more potent inhibitors. Structural modeling revealed that phosphorylation of AtPPI-2 induces conformational changes that modulate TOPP binding.