Complex Phosphorylation Pattern Research Articles

Hsc70 phosphorylation patterns and calmodulin regulate AP2 Clathrin-Coated-Vesicle life span for cell adhesion protein transport

AP2 forms AP2 CCV with clathrin and over 60 additional coat proteins. Due to this complexity, we have a limited understanding of CCV life cycle regulation. Synapses contain canonical AP2 CCV, canCCV, and more stable, thereby longer lived, AP2 CCV. The more stable AP2 CCV can be distinguished from canCCV due to the stable binding of Hsc70 to clathrin. The AP1/σ1B complex knockout leads to impaired synaptic vesicle recycling and altered endosomal protein sorting. This causes as a secondary phenotype the twofold upregulation of endocytosis by canCCV and by more stable AP2 CCV. These stable CCV are more stabilized than their wt counterpart, hence stCCV. They have less of the uncoating proteins synaptojanin1 and Hsc70, and more of the coat stabilizing AAK1. Hsc70 clathrin dissociation activity is regulated by complex phosphorylation patterns. Two major groups of hyper- and of hypo-phosphorylated Hsc70 proteins are formed. The latter are enriched in wt stable CCV and stabilized stCCV. Hsc70 T265 phosphorylation regulates binding of CaM/Ca2+. CaM/Ca2+ binding to the T265 domain blocks Hsc70 homodimerization and its concentration in stCCV required for clathrin disassembly. Kinases DYRK1A and CaMK-IIδ can phosphorylate T265 preventing CaM/Ca2+ binding. Their and the levels of STK38L and STK39/Cab39, which are able to phosphorylate additional Hsc70 residues are reduced in stCCV. The stCCV pathway sorts specifically the cell adhesion proteins CHL1 and Neurocan, supporting our model of that the stCCV pathway fulfills specific functions in synaptic plasticity.

Evaluating Spatiotemporal Dynamics of Phosphorylation of RNA Polymerase II Carboxy-Terminal Domain by Ultraviolet Photodissociation Mass Spectrometry.

The critical role of site-specific phosphorylation in eukaryotic transcription has motivated efforts to decipher the complex phosphorylation patterns exhibited by the carboxyl-terminal domain (CTD) of RNA polymerase II. Phosphorylation remains a challenging post-translational modification to characterize by mass spectrometry owing to the labile phosphate ester linkage and low stoichiometric prevalence, two features that complicate analysis by high-throughput MS/MS methods. Identifying phosphorylation sites represents one significant hurdle in decrypting the CTD phosphorylation, a problem exaggerated by a large number of potential phosphorylation sites. An even greater obstacle is decoding the dynamic phosphorylation pattern along the length of the periodic CTD sequence. Ultraviolet photodissociation (UVPD) is a high-energy ion activation method that provides ample backbone cleavages of peptides while preserving labile post-translational modifications that facilitate their confident localization. Herein, we report a quantitative parallel reaction monitoring (PRM) method developed to monitor spatiotemporal changes in site-specific Ser5 phosphorylation of the CTD by cyclin-dependent kinase 7 (CDK7) using UVPD for sequence identification, phosphosite localization, and differentiation of phosphopeptide isomers. We capitalize on the series of phospho-retaining fragment ions produced by UVPD to create unique transition lists that are pivotal for distinguishing the array of phosphopeptides generated from the CTD.

Four Phosphates at One Blow: Access to Pentaphosphorylated Magic Spot Nucleotides and Their Analysis by Capillary Electrophoresis.

The complex phosphorylation pattern of natural and modified pentaphosphorylated magic spot nucleotides is generated in a highly efficient way. A cyclic pyrophosphoryl phosphoramidite (cPyPA) reagent is used to introduce four phosphates on nucleosides regioselectively in a one-flask key transformation. The obtained magic spot nucleotides are used to develop a capillary electrophoresis UV detection method, enabling nucleotide assignment in complex bacterial extracts.

Regulation of Light Harvesting in Chlamydomonas reinhardtii Two Protein Phosphatases Are Involved in State Transitions.

Protein phosphorylation plays important roles in short-term regulation of photosynthetic electron transfer, and during state transitions, the kinase STATE TRANSITION7 (STT7) of Chlamydomonas reinhardtii phosphorylates components of light-harvesting antenna complex II (LHCII). This reversible phosphorylation governs the dynamic allocation of a part of LHCII to PSI or PSII, depending on light conditions and metabolic demands, but counteracting phosphatase(s) remain unknown in C. reinhardtii Here we analyzed state transitions in C. reinhardtii mutants of two phosphatases, PROTEIN PHOSPHATASE1 and PHOTOSYSTEM II PHOSPHATASE, which are homologous to proteins that antagonize the state transition kinases (STN7 and STN8) in Arabidopsis (Arabidopsis thaliana). The transition from state 2 to state 1 was retarded in pph1, and surprisingly also in pbcp However, both mutants eventually returned to state 1. In contrast, the double mutant pph1;pbcp appeared strongly locked in state 2. The complex phosphorylation patterns of the LHCII trimers and of the monomeric subunits were affected in the phosphatase mutants. Their analysis indicated that the two phosphatases have different yet overlapping sets of protein targets. The dual control of thylakoid protein dephosphorylation and the more complex antenna phosphorylation patterns in C. reinhardtii compared to Arabidopsis are discussed in the context of the stronger amplitude of state transitions and the more diverse LHCII isoforms in the alga.

Phosphorylation of the multifunctional signal transducer B-cell adaptor protein (BCAP) promotes recruitment of multiple SH2/SH3 proteins including GRB2

B-cell adaptor protein (BCAP) is a multimodular, multifunctional signal transducer that regulates signal transduction pathways in leukocytes, including macrophages, B-cells, and T-cells. In particular, BCAP suppresses inflammatory signaling by Toll-like receptors (TLRs). However, how BCAP itself is regulated and what its interaction partners are is unclear. Here, using human immune cell lines, including THP-1 cells, we characterized the complex phosphorylation patterns of BCAP and used a novel protein complex trapping strategy, called virotrap, to identify its interaction partners. This analysis identified known interactions of BCAP with phosphoinositide 3-kinase (PI3K) p85 subunit and NCK adaptor protein (NCK), together with previously unknown interactions of BCAP with Src homology 2 (SH2) and SH3 domain-containing adaptor proteins, notably growth factor receptor-bound protein 2 (GRB2) and CRK-like proto-oncogene, adaptor protein (CRKL). We show that the SH3 domain of GRB2 can bind to BCAP independently of BCAP phosphorylation status, suggesting that the SH2 domains mediate interactions with activated receptor tyrosine kinase complexes including the CD19 subunit of the B-cell receptor. Our results also suggested that the PI3K p85 subunit binds to BCAP via SH3 domains forming an inactive complex that is then activated by sequential binding with the SH2 domains. Taken together, our results indicate that BCAP is a complex hub that processes signals from multiple pathways in diverse cell types of the immune system.

The Complex Phosphorylation Patterns that Regulate the Activity of Hsp70 and Its Cochaperones.

Proteins must fold into their native structure and maintain it during their lifespan to display the desired activity. To ensure proper folding and stability, and avoid generation of misfolded conformations that can be potentially cytotoxic, cells synthesize a wide variety of molecular chaperones that assist folding of other proteins and avoid their aggregation, which unfortunately is unavoidable under acute stress conditions. A protein machinery in metazoa, composed of representatives of the Hsp70, Hsp40, and Hsp110 chaperone families, can reactivate protein aggregates. We revised herein the phosphorylation sites found so far in members of these chaperone families and the functional consequences associated with some of them. We also discuss how phosphorylation might regulate the chaperone activity and the interaction of human Hsp70 with its accessory and client proteins. Finally, we present the information that would be necessary to decrypt the effect that post-translational modifications, and especially phosphorylation, could have on the biological activity of the Hsp70 system, known as the “chaperone code”.

Sites phosphorylated in human α1B-adrenoceptors in response to noradrenaline and phorbol myristate acetate

Phosphorylation of the human α1B-adrenergic receptor (fused with the green fluorescent protein) was studied employing the inducible Flp-ln HEK293 T-Rex system for expression. Serine/alanine substitutions were performed in five sites corresponding to those previously identified as phosphorylation targets in the hamster ortholog. Desensitization was decreased in these mutants but receptor phosphorylation was still clearly detected. The protein phosphorylation of the wild-type receptor (fused to the green fluorescent protein) was studied, using mass spectrometry, under baseline and stimulated conditions (noradrenaline or phorbol myristate acetate). Basal phosphorylation was detected at sites located at the intracellular loop 3 and carboxyl terminus, and the number of sites detected increased under agonist activation and stimulation of protein kinase C. The phosphorylation patterns differed under the distinct conditions. Three of the phosphorylation sites detected in this work corresponded to those observed in the hamster receptor. The phosphorylation sites detected included the following: a) at the intracellular loop 3: serines 246, 248, 257, 267, and 277; and threonines 252, 264, and 268, and b) at the carboxyl terminus: serines 396, 400, 402, 406, 423, 425, 427, 455, and 470, and threonines 387, 392, 420, and 475. Our data indicate that complex phosphorylation patterns exist and suggest the possibility that such differences could be relevant in receptor function and subcellular localization.

Aging dependent effect of nuclear tau

Tau protein is characterized by a complex pattern of phosphorylation and is localized in the cytoplasm and nucleus in both neuronal and non-neuronal cells. Human AT100 nuclear tau, endowed by phosphorylation in Thr212/Ser214, was recently shown to decline in cornus ammonis 1 (CA1) and dentate gyrus (DG) in Alzheimer’s disease (AD), but a defined function for this nuclear tau remains unclear. Here we show that AT100 progressively increases in the nuclei of neuronal and non-neuronal cells during aging, and decreases in the more severe AD stages, as recently shown, and in cancer cells (colorectal adenocarcinoma and breast cancer). AT100, in addition to a co-localization with the DAPI-positive heterochromatin, was detected in the nucleolus of pyramidal cells from the CA1 region, shown to be at its highest level in the more senescent cells and in the first stage of AD (ADI), and disappearing in the more severe AD cases (ADIV). Taking into account the nuclear distribution of AT100 during cell aging and its relation to the chromatin changes observed in degenerated neurons, as well as in cancerous cells, which are both cellular pathologies associated with age, we can consider the Thr212/Ser214 phosphorylated nuclear tau as a molecular marker of cell aging.

Neurofilaments and Neurofilament Proteins in Health and Disease.

SUMMARYNeurofilaments (NFs) are unique among tissue-specific classes of intermediate filaments (IFs) in being heteropolymers composed of four subunits (NF-L [neurofilament light]; NF-M [neurofilament middle]; NF-H [neurofilament heavy]; and α-internexin or peripherin), each having different domain structures and functions. Here, we review how NFs provide structural support for the highly asymmetric geometries of neurons and, especially, for the marked radial expansion of myelinated axons crucial for effective nerve conduction velocity. NFs in axons extensively cross-bridge and interconnect with other non-IF components of the cytoskeleton, including microtubules, actin filaments, and other fibrous cytoskeletal elements, to establish a regionally specialized network that undergoes exceptionally slow local turnover and serves as a docking platform to organize other organelles and proteins. We also discuss how a small pool of oligomeric and short filamentous precursors in the slow phase of axonal transport maintains this network. A complex pattern of phosphorylation and dephosphorylation events on each subunit modulates filament assembly, turnover, and organization within the axonal cytoskeleton. Multiple factors, and especially turnover rate, determine the size of the network, which can vary substantially along the axon. NF gene mutations cause several neuroaxonal disorders characterized by disrupted subunit assembly and NF aggregation. Additional NF alterations are associated with varied neuropsychiatric disorders. New evidence that subunits of NFs exist within postsynaptic terminal boutons and influence neurotransmission suggests how NF proteins might contribute to normal synaptic function and neuropsychiatric disease states.

Mutation of Light-dependent Phosphorylation Sites of the Drosophila Transient Receptor Potential-like (TRPL) Ion Channel Affects Its Subcellular Localization and Stability

The Drosophila phototransduction cascade terminates in the opening of the ion channel transient receptor potential (TRP) and TRP-like (TRPL). Contrary to TRP, TRPL undergoes light-dependent subcellular trafficking between rhabdomeric photoreceptor membranes and an intracellular storage compartment, resulting in long term light adaptation. Here, we identified in vivo phosphorylation sites of TRPL that affect TRPL stability and localization. Quantitative mass spectrometry revealed a light-dependent change in the TRPL phosphorylation pattern. Mutation of eight C-terminal phosphorylation sites neither affected multimerization of the channels nor the electrophysiological response of flies expressing the mutated channels. However, these mutations resulted in mislocalization and enhanced degradation of TRPL after prolonged dark-adaptation. Mutation of subsets of the eight C-terminal phosphorylation sites also led to a reduction of TRPL content and partial mislocalization in the dark. This suggests that a light-dependent switch in the phosphorylation pattern of the TRPL channel mediates stable expression of TRPL in the rhabdomeres upon prolonged dark-adaptation.

Change of carbon source causes dramatic effects in the phospho-proteome of the archaeon Sulfolobus solfataricus.

Protein phosphorylation is known to occur in Archaea. However, knowledge of phosphorylation in the third domain of life is rather scarce. Homology-based searches of archaeal genome sequences reveals the absence of two-component systems in crenarchaeal genomes but the presence of eukaryotic-like protein kinases and protein phosphatases. Here, the influence of the offered carbon source (glucose versus tryptone) on the phospho-proteome of Sulfolobus solfataricus P2 was studied by precursor acquisition independent from ion count (PAcIFIC). In comparison to previous phospho-proteome studies, a high number of phosphorylation sites (1318) located on 690 phospho-peptides from 540 unique phospho-proteins were detected, thus increasing the number of currently known archaeal phospho-proteins from 80 to 621. Furthermore, a 25.8/20.6/53.6 Ser/Thr/Tyr percentage ratio with an unexpectedly high predominance of tyrosine phosphorylation was detected. Phospho-proteins in most functional classes (21 out of 26 arCOGs) were identified, suggesting an important regulatory role in S. solfataricus. Focusing on the central carbohydrate metabolism in response to the offered carbon source, significant changes were observed. The observed complex phosphorylation pattern hints at an important physiological function of protein phosphorylation in control of the central carbohydrate metabolism, which might particularly operate in channeling carbon flux into the respective metabolic pathways.

7-Nitroindazole down-regulates dopamine/DARPP-32 signaling in neostriatal neurons in a rat model of Parkinson's disease

Neuronal nitric oxide synthase (nNOS) is involved in the regulation of diverse intracellular messenger systems in the brain. Nitric Oxide (NO) contributes to inducing signaling cascades that involve a complex pattern of phosphorylation of DARPP-32 (in Thr-34), which controls the phosphoproteins involved in neuronal activation. However, the role of NO in the pathophysiology of Parkinson's disease (PD) and its effect in striatal neurons have been scarcely explored. In the present work, we investigate the effects of a nitric oxide synthase (NOS) inhibitor, 7-nitroindazole (7-NI) in the nigrostriatal pathway of striatal 6-hydroxydopamine (6-OHDA) lesioned rats. Our quantitative histological findings show that treatment with 7-NI significantly reduced 6-OHDA-induced dopaminergic damage in the dorsolateral striatum and Substantia Nigra pars compacta (SNpc). Moreover, 6-OHDA lesioned rats show a significant increase of nNOS+ and Phospho-Thr34-DARPP-32+ cells, accompanied by a consequent decrease of total DARPP-32+ cells, which suggests an imbalance of NO activity in the DA-depleted striatum, which is also reflected in behavioral studies. Importantly, these effects are reverted in the group treated with 7-NI. These results show a clear link between the state of phosphorylation of DARPP-32 and parkinsonism, which is regulated by nNOS. This new evidence suggests a prominent role for nitric oxide in the neurotransmitter balance within the basal ganglia in the pathophysiology of experimental parkinsonism.

Sequential Multiplex Analyte Capturing for Phosphoprotein Profiling

Microarray-based sandwich immunoassays can simultaneously detect dozens of proteins. However, their use in quantifying large numbers of proteins is hampered by cross-reactivity and incompatibilities caused by the immunoassays themselves. Sequential multiplex analyte capturing addresses these problems by repeatedly probing the same sample with different sets of antibody-coated, magnetic suspension bead arrays. As a miniaturized immunoassay format, suspension bead array-based assays fulfill the criteria of the ambient analyte theory, and our experiments reveal that the analyte concentrations are not significantly changed. The value of sequential multiplex analyte capturing was demonstrated by probing tumor cell line lysates for the abundance of seven different receptor tyrosine kinases and their degree of phosphorylation and by measuring the complex phosphorylation pattern of the epidermal growth factor receptor in the same sample from the same cavity.

DDK Phosphorylates Checkpoint Clamp Component Rad9 and Promotes Its Release from Damaged Chromatin

When inappropriate DNA structures arise, they are sensed by DNA structure-dependent checkpoint pathways and subsequently repaired. Recruitment of checkpoint proteins to such structures precedes recruitment of proteins involved in DNA metabolism. Thus, checkpoints can regulate DNA metabolism. We show that fission yeast Rad9, a 9-1-1 heterotrimeric checkpoint-clamp component, is phosphorylated by Hsk1(Cdc7), the Schizosaccharomyces pombeDbf4-dependent kinase (DDK) homolog, in response to replication-induced DNA damage. Phosphorylation of Rad9 disrupts its interaction with replication protein A (RPA) and is dependent on 9-1-1 chromatin loading, the Rad9-associated protein Rad4/Cut5(TopBP1), and prior phosphorylation by Rad3(ATR). rad9 mutants defective in DDK phosphorylation show wild-type checkpoint responses but abnormal DNA repair protein foci and decreased viability after replication stress. We propose that Rad9 phosphorylation by DDK releases Rad9 from DNA damage sites to facilitate DNA repair.

Homologous desensitization of guanylyl cyclase A, the receptor for atrial natriuretic peptide, is associated with a complex phosphorylation pattern

Atrial natriuretic peptide (ANP), via its guanylyl cyclase A (GC-A) receptor and intracellular guanosine 3′,5′-cyclic monophosphate production, is critically involved in the regulation of blood pressure. In patients with chronic heart failure, the plasma levels of ANP are increased, but the cardiovascular actions are severely blunted, indicating a receptor or postreceptor defect. Studies on metabolically labelled GC-A-overexpressing cells have indicated that GC-A is extensively phosphorylated, and that ANP-induced homologous desensitization of GC-A correlates with receptor dephosphorylation, a mechanism which might contribute to a loss of function in vivo. In this study, tandem MS analysis of the GC-A receptor, expressed in the human embryonic kidney cell line HEK293, revealed unambiguously that the intracellular domain of the receptor is phosphorylated at multiple residues: Ser487, Ser497, Thr500, Ser502, Ser506, Ser510 and Thr513. MS quantification based on multiple reaction monitoring demonstrated that ANP-provoked desensitization was accompanied by a complex pattern of receptor phosphorylation and dephosphorylation. The population of completely phosphorylated GC-A was diminished. However, intriguingly, the phosphorylation of GC-A at Ser487 was selectively enhanced after exposure to ANP. The functional relevance of this observation was analysed by site-directed mutagenesis. The substitution of Ser487 by glutamate (which mimics phosphorylation) blunted the activation of the GC-A receptor by ANP, but prevented further desensitization. Our data corroborate previous studies suggesting that the responsiveness of GC-A to ANP is regulated by phosphorylation. However, in addition to the dephosphorylation of the previously postulated sites (Ser497, Thr500, Ser502, Ser506, Ser510), homologous desensitization seems to involve the phosphorylation of GC-A at Ser487, a newly identified site of phosphorylation. The identification and further characterization of the specific mechanisms involved in the downregulation of GC-A responsiveness to ANP may have important pathophysiological implications.Structured digital abstract•MINT-7713870, MINT-7713887: PMCA (uniprotkb:P20020) and GC-A (uniprotkb:P18910) colocalize (MI:0403) by fluorescence microscopy (MI:0416)

Analysis of rotavirus non-structural protein NSP5 by mass spectrometry reveals a complex phosphorylation pattern

Genomic replication and partial assembly of Rotavirus takes place in cytoplasmic viral structures called viroplasms. NSP5 is a viral phosphoprotein localized in viroplasms and its expression is imperative for viral cycle progress. During infection three isoforms of NSP5 can be observed by SDS-PAGE (26, 28 and 33–35 kDa) and previous reports suggested that they differ in their phosphorylation patterns. In this study we obtained NSP5 from infected cells and by mass spectrometry we were able to identify nine phosphorylation sites. We detected that in all the isoforms the same residues can be found either phosphorylated or unmodified. Quantitative analysis showed that the 28 kDa isoform has a higher phosphorylation level than the 26 kDa isoform suggesting that migration properties depend on the total number of phosphorylated residues. Moreover, we identified two not previously described modifications for this protein: an N-acetylation in Serine-2 and an intramolecular disulfide bond in a highly conserved motif, CXXC which is located between two charged alpha-helix motifs.

Phosphorylation of Human Insulin Receptor Substrate-1 at Serine 629 Plays a Positive Role in Insulin Signaling

The function of insulin receptor substrate-1 (IRS-1) is regulated by both tyrosine and serine/threonine phosphorylation. Phosphorylation of some serine/threonine residues in IRS-1 dampens insulin signaling, whereas phosphorylation of other serine/threonine residues enhances insulin signaling. Phosphorylation of human IRS-1 at Ser(629) was increased by insulin in Chinese hamster ovary cells expressing the insulin receptor (1.26 +/- 0.09-fold; P < 0.05) and L6 cells (1.35 +/- 0.29-fold; P < 0.05) expressing human IRS-1. Sequence analysis surrounding Ser(629) revealed conformity to the consensus phosphorylation sequence recognized by Akt. Phosphorylation of IRS-1 at Ser(629) in cells was decreased upon treatment with either an Akt inhibitor or by coexpression with kinase dead Akt, whereas Ser(629) phosphorylation was increased by coexpression with constitutively active Akt. In addition, Ser(629) of IRS-1 is directly phosphorylated by Akt in vitro. In cells, preventing phosphorylation of Ser(629) by a Ser(629)Ala mutation resulted in increased phosphorylation of Ser(636), a known negative regulator of IRS-1, without affecting phosphorylation of Tyr(632) or Ser(616). Cells expressing the Ser(629)Ala mutation, along with increased Ser(636) phosphorylation, had decreased insulin-stimulated association of the p85 regulatory subunit of phosphatidylinositol 3'-kinase with IRS-1 and decreased phosphorylation of Akt at Ser(473). Finally, in vitro phosphorylation of a Ser(629)-containing IRS-1 fragment with Akt reduces the subsequent ability of ERK to phosphorylate Ser(636/639). These results suggest that a feed-forward mechanism may exist whereby insulin activation of Akt leads to phosphorylation of IRS-1 at Ser(629), resulting in decreased phosphorylation of IRS-1 at Ser(636) and enhanced downstream signaling. Understanding the complex phosphorylation patterns of IRS-1 is crucial to elucidating the factors contributing to insulin resistance and, ultimately, the pathogenesis of type 2 diabetes.

High osmolarity glycerol (HOG) pathway-induced phosphorylation and activation of 6-phosphofructo-2-kinase are essential for glycerol accumulation and yeast cell proliferation under hyperosmotic stress.

In response to changes in the environment, yeast cells coordinate intracellular activities to optimize survival and proliferation. The transductions of diverse extracellular stimuli are exerted through multiple mitogen-activated protein kinase (MAPK) cascades. The high osmolarity glycerol (HOG) MAPK pathway is activated by increased environmental osmolarity and results in a rise of the cellular glycerol concentration to adapt the intracellular osmotic pressure. We studied the importance of the short time regulation of glycolysis under hyperosmotic stress for the survival and proliferation of yeast cells. A stimulation of the HOG-MAPK pathway by increasing the medium osmolarity through addition of salt or glucose to cultivated yeast leads to an activation of 6-phosphofructo-2-kinase (PFK2), which is accompanied by a complex phosphorylation pattern of the enzyme. An increase in medium osmolarity with 5% NaCl activates PFK2 3-fold over the initial value. This change in the activity is the result of a 4-fold phosphorylation of the enzyme mediated by protein kinases from the HOG-MAPK pathway. In the case of hyperosmolar glucose a 5-fold PFK2 activation was achieved by a single phosphorylation with protein kinase A near the carboxyl terminus of the protein on Ser(644) and an additional 5-fold phosphorylation within the same amino-terminal fragment as in the presence of salt. The effect of hyperosmolar glucose is the result of an activation of the Ras-cAMP pathway together with the HOG-MAPK pathway. The activation of PFK2 leads to an activation of the upper part of glycolysis, which is a precondition for glycerol accumulation. Yeast cells containing PFK2 accumulate three times more glycerol than cells lacking PFK2, which are not able to grow under hypertonic stress.

Phosphorylation of calcipressin 1 increases its ability to inhibit calcineurin and decreases calcipressin half-life.

Calcipressin 1 is an endogenous inhibitor of calcineurin, which is a serine/threonine phosphatase under the control of Ca(2+) and calmodulin. Calcipressin 1 is encoded by DSCR1, a gene on human chromosome 21 with seven exons, exons 1-4 are alternative first exons (isoforms 1-4). We show that calcipressin 1 isoform 1 has an N-terminal coding region longer than that previously described, and this generates a new polypeptide of 252 amino acids. This polypeptide is able to interact with calcineurin A and to inhibit NF-AT-mediated transcriptional activation. We demonstrate for the first time that endogenous calcipressin 1 exists as a complex together with the calcineurin A and B heterodimer. Calcipressin 1 is a phosphoprotein that increases its capacity to inhibit calcineurin when phosphorylated at the FLISPP motif, and this phosphorylation also controls the half-life of calcipressin 1 by accelerating its degradation. Additionally, we have also detected further phosphorylation sites outside the FLISPP motif and these contribute to the complex phosphorylation pattern of calcipressin 1. Taking all these results into consideration we suggest that phosphorylation of calcipressin 1 is involved in the regulation of the phosphatase activity of calcineurin and can therefore act as a modulator of calcineurin-dependent cellular pathways.

Glucose-induced stimulation of the Ras-cAMP pathway in yeast leads to multiple phosphorylations and activation of 6-phosphofructo-2-kinase.

Yeast cells respond to changes of the environment by complex modifications of the metabolism. An increase of the extracellular glucose concentration activates the Ras-cAMP pathway. Via a production of cAMP this pathway stimulates the cAMP-dependent protein kinase (PKA) which is involved in the posttranslational regulation of the key enzymes of gluconeogenesis and glycolysis. 6-Phosphofructo-2-kinase (PFK2) catalyzes the synthesis of fructose 2,6-bisphosphate, the most potent activator of the glycolytic key enzyme 6-phosphofructo-1-kinase. We investigated the molecular mechanism of the glucose-induced phosphorylation and activation of PFK2 in Saccharomyces cerevisiae. After an incubation of PFK2 with ATP and PKA in vitro, two amino acid residues, Thr157 and Ser644, are phosphorylated and the enzyme is activated. A stimulation of the Ras-cAMP pathway by glucose addition to cultivated yeast cells leads to an in vivo activation of PFK2 which is accompanied by a more complex phosphorylation pattern of the enzyme. The phosphorylation of the protein on Ser644 is the result of PKA stimulation while the protein kinase(s) catalyzing the 5-fold phosphorylation of the peptide fragment T(67)(-)(101) is (are) still unknown. The functional significance of T(67)(-)(101) and its phosphorylation is supported by the finding that PFK2 lacking this peptide is inactive.