An Arabidopsis protein phosphorylated in response to microbial elicitation, AtPHOS32, is a substrate of MAP kinases 3 and 6

Although mitogen-activated protein kinases (MAPKs) have been shown to be activated by a wide range of biotic and abiotic stimuli in diverse plant species, few in vivo substrates for these kinases have been identified. While studying proteins that are differentially phosphorylated upon treatment of Arabidopsis suspension cultures with the general bacterial elicitor peptide flagellin-22 (flg22), we identified two proteins with endogenous nickel binding properties that become phosphorylated after flg22 elicitation. These highly related proteins, AtPHOS32 and AtPHOS34, show similarity to bacterial universal stress protein A. We identified one of the phosphorylation sites on AtPHOS32 by nanoelectrospray ionization tandem mass spectrometry. Phosphorylation in a phosphoSer-Pro motif indicated that this protein may be a substrate of MAPKs. Using in vitro kinase assays, we confirmed that AtPHOS32 is a substrate of both AtMPK3 and AtMPK6. Specificity of phosphorylation was demonstrated by site-directed mutagenesis of the first phosphorylation site. In addition, immunosubtraction of both MAPKs from protein extracts removed detectable kinase activity toward AtPHOS32, indicating that the two MAPKs were the predominate kinases recognizing the motif in this protein. Finally, the target phosphorylation site in AtPHOS32 is conserved in AtPHOS34 and among apparent orthologues from many plant species, indicating that phosphorylation of these proteins by AtMPK3 and AtMPK6 orthologues has been conserved throughout evolution.     

Phosphorylation dynamics of membrane proteins from Arabidopsis roots submitted to salt stress

An excess of NaCl in the soil is detrimental for plant growth. It interferes with mineral nutrition and water uptake and leads to accumulation of toxic ions in the plant. Understanding the response of roots to NaCl stress may facilitate the development of crops with increased tolerance to this and other stresses. Since controls achieved at the posttranslational level are of critical importance for regulating protein function, the present work used a robust label‐free quantitative proteomic methodology to quantify phosphorylation events that affect root membrane proteins in Arabidopsis, in response to short‐term (up to 2 h) NaCl treatments. This work identified 302 proteotypic phosphopeptides including 77 novel phosphorylated sites. NaCl treatment significantly altered the abundance of 74 phosphopeptides, giving novel insights into the regulation of major classes of membrane proteins, including ATPases, sodium transporters, and aquaporins. The data provide a unique access to phosphorylation reprogramming of ionic equilibrium in plant cells under NaCl stress. The use of predictive bioinformatic tools for kinase motifs suggested that root membrane proteins are substrates of cAMP‐dependent protein kinase, cGMP‐dependent protein kinase, and protein kinase C families, also called AGC kinases, arguing for an important role of lipid signaling in abiotic stress responses. It also pointed to cross‐talks between protein kinase families during NaCl stress.     

Proteomic analysis of in vivo phosphorylated synaptic proteins

In the nervous system, protein phosphorylation is an essential feature of synaptic function. Although protein phosphorylation is known to be important for many synaptic processes and in disease, little is known about global phosphorylation of synaptic proteins. Heterogeneity and low abundance make protein phosphorylation analysis difficult, particularly for mammalian tissue samples. Using a new approach, combining both protein and peptide immobilized metal affinity chromatography and mass spectrometry data acquisition strategies, we have produced the first large scale map of the mouse synapse phosphoproteome. We report over 650 phosphorylation events corresponding to 331 sites (289 have been unambiguously assigned), 92% of which are novel. These represent 79 proteins, half of which are novel phosphoproteins, and include several highly phosphorylated proteins such as MAP1B (33 sites) and Bassoon (30 sites). An additional 149 candidate phosphoproteins were identified by profiling the composition of the protein immobilized metal affinity chromatography enrichment. All major synaptic protein classes were observed, including components of important pre- and postsynaptic complexes as well as low abundance signaling proteins. Bioinformatic and in vitro phosphorylation assays of peptide arrays suggest that a small number of kinases phosphorylate many proteins and that each substrate is phosphorylated by many kinases. These data substantially increase existing knowledge of synapse protein phosphorylation and support a model where the synapse phosphoproteome is functionally organized into a highly interconnected signaling network.     

c-Jun N-terminal kinase-3 (JNK3)/stress-activated protein kinase-beta (SAPKbeta) binds and phosphorylates the neuronal microtubule regulator SCG10.

The neuronal growth-associated protein SCG10 is enriched in the growth cones of neurons where it destabilizes microtubules and thus contributes to the dynamic assembly and disassembly of microtubules. Since its microtubule-destabilizing activity is regulated by phosphorylation, SCG10 may link extracellular signals to rearrangements of the neuronal cytoskeleton. To identify signal transduction pathways that may lead to SCG10 phosphorylation, we tested a series of serine-threonine-directed protein kinases that phosphorylate SCG10 in vitro. We demonstrate that purified SCG10 can be phosphorylated by two subclasses of mitogen-activated protein (MAP) kinases, c-Jun N-terminal/stress-activated protein kinase (JNK/SAPK) and p38 MAP kinase. Moreover, SCG10 was found to bind tightly and specifically to JNK3/SAPKbeta. JNK3/SAPKbeta phosphorylation occurs at Ser-62 and Ser-73, residues that result in reduced microtubule-destabilizing activity for SCG10. Endogenous SCG10 also undergoes increased phosphorylation in sympathetic neurons at times of JNK3/SAPKbeta activation following deprivation from nerve growth factor. Together these observations indicate that activation of JNK/SAPKs provides a pathway for phosphorylation of SCG10 and control of growth cone microtubule formation following neuronal exposure to cellular stresses.     

Identification of novel interactors and potential phosphorylation substrates of GsSnRK1 from wild soybean (Glycine soja)

The plant sucrose nonfermenting kinase 1 (SnRK1) kinases play the central roles in the processes of energy balance, hormone perception, stress resistance, metabolism, growth, and development. However, the functions of these kinases are still elusive. In this study, we used GsSnRK1 of wild soybean as bait to perform library‐scale screens by the means of yeast two‐hybrid to identify its interacting proteins. The putative interactions were verified by yeast retransformation and β‐galactosidase assays, and the selected interactions were further confirmed in planta by bimolecular fluorescence complementation and biochemical Co‐IP assays. Protein phosphorylation analyses were carried out by phos‐tag assay and anti‐phospho‐(Ser/Thr) substrate antibodies. Finally, we obtained 24 GsSnRK1 interactors and several putative substrates that can be categorized into SnRK1 regulatory β subunit, protein modification, biotic and abiotic stress‐related, hormone perception and signalling, gene expression regulation, water and nitrogen transport, metabolism, and unknown proteins. Intriguingly, we first discovered that GsSnRK1 interacted with and phosphorylated the components of soybean nodulation and symbiotic nitrogen fixation. The interactions and potential functions of GsSnRK1 and its associated proteins were extensively discussed and analysed. This work provides plausible clues to elucidate the novel functions of SnRK1 in response to variable environmental, metabolic, and physiological requirements.     

Hyperphosphorylation of JNK-interacting protein 1, a protein associated with Alzheimer disease

The c-Jun N-terminal kinase (JNK) group of mitogen-activated protein (MAP) kinases are activated by pleiotropic signals including environmental stresses, growth factors, and hormones. JNK-interacting protein 1 (JIP1) is a scaffold protein that assembles and facilitates the activation of the mixed lineage kinase-dependent JNK module and also establishes an interaction with beta-amyloid precursor protein that has been partially characterized. Here we show that, similarly to other proteins involved in various neurological diseases, JIP1 becomes hyperphosphorylated following activation of stress-activated and MAP kinases. By immobilized metal affinity chromatography and a combined microcapillary LC/MALDI-TOF/ESI-ion trap mass spectrometry approach, we identified 35 sites of mitotic phosphorylation within JIP1, among which eight were present within (Ser/Thr)-Pro sequence. This motif is modified by various kinases in aggregates of the microtubule-associated protein tau, which generates typical intraneuronal lesions occurring in Alzheimer disease. Most of the post-translational modifications found were located within the JNK, MAP kinase kinase, and RAC-alpha Ser/Thr protein kinase binding regions; no modifications occurred in protein Src homology 3 and phosphotyrosine interaction domains, which are essential for binding to kinesin, beta-amyloid precursor protein, and MAP kinase kinase kinase. Protein phosphorylation is known to affect stability and protein-protein interactions. Thus, the findings that JIP1 is extensively phosphorylated after activation of stress-activated and MAP kinases indicate that these signaling pathways might modulate JIP1 signaling by regulating its stability and association with some, but not all, interacting proteins.     

Phosphorylation sites on tau identified by nanoelectrospray mass spectrometry: differences in vitro between the mitogen-activated protein kinases ERK2, c-Jun N-terminal kinase and P38, and glycogen synthase kinase-3beta

The stress-activated kinases c-Jun N-terminal kinase (JNK) and p38 are members of the mitogen-activated protein (MAP) kinase family and take part in signalling cascades initiated by various forms of stress. Their targets include the microtubule-associated protein tau, which becomes hyperphosphorylated in Alzheimer's disease. It is necessary, as a forerunner for in vivo studies, to identify the protein kinases and phosphatases that are responsible for phosphate turnover at individual sites. Using nanoelectrospray mass spectrometry, we have undertaken an extensive comparison of phosphorylation in vitro by several candidate tau kinases, namely, JNK, p38, ERK2, and glycogen synthase kinase 3beta (GSK3beta). Between 10 and 15 sites were identified for each kinase. The three MAP kinases phosphorylated Ser202 and Thr205 but not detectably Ser199, whereas conversely GSK3beta phosphorylated Ser199 but not detectably Ser202 or Thr205. Phosphorylated Ser404 was found with all of these kinases except JNK. The MAP kinases may not be strictly proline specific: p38 phosphorylated the nonproline sites Ser185, Thr245, Ser305, and Ser356, whereas ERK2 was the most strict. All of the sites detected except Thr245 and Ser305 are known or suspected phosphorylation sites in paired helical filament-tau extracted from Alzheimer brains. Thus, the three MAP kinases and GSK3beta are importantly all strong candidates as tau kinases that may be involved in the pathogenic hyperphosphorylation of tau in Alzheimer's disease.     

Roles of “junk phosphorylation” in modulating biomolecular association of phosphorylated proteins?

Protein phosphorylation dynamically regulates cellular activities in response to environmental cues. Sequence conservation analysis of recent proteome-wide phosphorylation data revealed that many previously unidentified phosphorylation sites are not well-conserved leading to the proposal that many are non-functional. However, this is based on the assumption that protein phosphorylation modulates protein function through specific position on protein sequence. Based on emerging understanding on phospho-regulation of cellular activities, we argue, with examples, that non-positionally conserved phosphorylation sites can very well be functional. We previously identified phosphorylation events that need not be conserved at same positions across orthologous proteins but are likely maintained by evolutionary conserved signaling networks through orthologous kinases. We found that proteins with such conserved phosphorylation patterns are statistically over-represented with protein and DNA-binding annotation. Here, we further correlated these proteins with protein-protein interaction data from an independent systematic study and observed they indeed interact frequently with other proteins. Hence, we speculate that non-positionally conserved phosphorylation site could be modulating biomolecular association of phosphorylated proteins possibly through fine-tuning protein’s bulk electrostatic charge and through creating binding sites for phospho-binding interaction domains. We, therefore, advocate the development of complementary evolutionary approaches to interpret physiological important sites.     

MAPK cascades and major abiotic stresses

Plants have evolved with complex signaling circuits that operate under multiple conditions and govern numerous cellular functions. Stress signaling in plant cells is a sophisticated network composed of interacting proteins organized into tiered cascades where the function of a molecule is dependent on the interaction and the activation of another. In a linear scheme, the receptors of cell surface sense the stimuli and convey stress signals through specific pathways and downstream phosphorylation events controlled by mitogen-activated protein (MAP) kinases and second messengers, leading to appropriate adaptive responses. The specificity of the pathway is guided by scaffolding proteins and docking domains inside the interacting partners with distinctive structures and functions. The flexibility and the fine-tuned organization of the signaling molecules drive the activated MAP kinases into the appropriate location and connection to control and integrate the information flow. Here, we overview recent findings of the involvement of MAP kinases in major abiotic stresses (drought, cold and temperature fluctuations) and we shed light on the complexity and the specificity of MAP kinase signaling modules.     

Different properties of SEK1 and MKK7 in dual phosphorylation of stress-induced activated protein kinase SAPK/JNK in embryonic stem cells

Stress-activated protein kinase/c-Jun NH(2)-terminal kinase (SAPK/JNK), belonging to the mitogen-activated protein kinase family, plays an important role in stress signaling. SAPK/JNK activation requires the phosphorylation of both Thr and Tyr residues in its Thr-Pro-Tyr motif, and SEK1 and MKK7 have been identified as the dual specificity kinases. In this study, we generated mkk7(-/-) mouse embryonic stem (ES) cells in addition to sek1(-/-) cells and compared the two kinases in terms of the activation and phosphorylation of JNK. Although SAPK/JNK activation by various stress signals was markedly impaired in both sek1(-/-) and mkk7(-/-) ES cells, there were striking differences in the dual phosphorylation profile. The severe impairment observed in mkk7(-/-) cells was accompanied by a loss of the Thr phosphorylation of JNK without marked reduction in its Tyr-phosphorylated level. On the other hand, Thr phosphorylation of JNK in sek1(-/-) cells was also attenuated in addition to a decreased level of its Tyr phosphorylation. Analysis in human embryonic kidney 293T cells transfected with a kinase-dead SEK1 or a Thr-Pro-Phe mutant of JNK1 revealed that SEK1-induced Tyr phosphorylation of JNK1 was followed by additional Thr phosphorylation by MKK7. Furthermore, SEK1 but not MKK7 was capable of binding to JNK1 in 293T cells. These results indicate that the Tyr and Thr residues of SAPK/JNK are sequentially phosphorylated by SEK1 and MKK7, respectively, in the stress-stimulated ES cells.     

5-HT induced accumulation of 3',5'-AMP and the phosphorylation of paramyosin in the ABRM of Mytilus edulis.

In Mytilus edulis 5-Ht induced relaxation of a muscle in catch is preceded by an increase in 3',5'-AMP content. In vitro two proteins of the contractile apparatus are phosphorylated by 3',5'-AMP dependent protein kinases. The 295000 dalton protein cannot be identified, the other one is paramyosin. Phosphorylated paramyosin inhibits actomyosin ATPase of smooth mollusc muscles at low and high Ca++ concentrations.     

Stress protein flux during recovery from simulated ischemia: induced heat shock protein 70 confers cytoprotection by suppressing JNK activation and inhibiting apoptotic cell death

Multiple stress proteins are recruited in response to stress in living cells. There are limited reports in the literature analyzing multiple stress protein shifts and their functional consequences on stress response. Using two-dimensional electrophoresis we have analyzed shifts in stress protein profiles in response to energy deprivation as a model of ischemic injury to kidneys. A group of chaperones and stress-induced mitogen activated protein (MAP) kinases were analyzed. In addition to examining stress protein induction and phosphorylation we have also examined the mechanism of cytoprotection by heat shock protein 70 (Hsp70). Our results show that, of the different stress proteins examined, only binding protein (BiP) and Hsp70 were significantly induced upon energy deprivation. Other stress proteins, including Hsp27, calnexin, Hsp90 and ERp57 showed alterations in their phosphorylation profiles. Three different MAP kinases, namely p38, extracellular signal regulated kisase and c-jun N-terminal kinase (JNK) were activated in response to energy deprivation. While JNK activation was linked to apoptosis, activated-p38 was involved in phosphorylation of Hsp27. Study of inhibitors of Hsp70 induction or pre-induction of Hsp70 indicated that induced Hsp70 was involved in the suppression of JNK activation thereby inhibiting apoptotic cell death. Our results provide important insights into the flux in stress protein profiles in response to simulated ischemia and highlight the antiapoptotic, cytoprotective mechanism of Hsp70 action.     

Proteomic and phosphoproteomic analyses of NaCl stress-responsive proteins in Arabidopsis roots

Salt is one of the major abiotic stresses limiting the productivity and the geographical distribution of crops. To gain a better understanding of NaCl stress responses in model plant Arabidopsis roots, the protein changes in the abundance (Coomassie Brilliant Blue R-350 stain) and phosphorylation (Pro-Q Diamond stain) were examined using two-dimensional electrophoresis coupled with mass spectrometry (MS). Seventeen unique proteins differentially changed in abundance, phosphorylation, or both in response to NaCl. Nonsynchronous differences were found between total proteins and phosphorylated proteins. Protein synthesis, proteolysis, post-translational modifications, and isoforms might cause the differential protein redundancies. The identified proteins are involved in binding, catalysis, signal transduction, transport, metabolisms of cell wall and energy, and reactive oxygen species (ROS) scavenging and defense. These protein changes provide new avenues of investigation into the underlying salt stress response in Arabidopsis roots and demonstrate the advantages of proteomic approach in plant biology studies.