Consensus Sequence For Phosphorylation Research Articles

Novel wiring of the AKT-RSK2 signaling pathway plays an essential role in cancer cell proliferation via a G1/S cell cycle transition

p90 Ribosomal S6 kinase 2 (RSK2), a member of mitogen-activated protein kinase regulating cell proliferation and transformation induced by tumor promoters, such as epidermal growth factor, plays a vital role as a signaling hub to modulate cell proliferation, transformation, cell cycle transition, and chromatin remodeling by tumor promoter stimulation such as epidermal growth factor. On the other hand, the RSK2-mediated signaling networks that regulate cancer cell proliferation are unclear. In this study, SKOV3, an ovarian cancer cell that exhibits chemoresistant properties, and TOV-112D cells showed different sensitivities to colony growth in soft agar. Based on the protein profile shown in a previous report, RSK2 knockdown preferentially and significantly suppressed cell proliferation and colony growth. Moreover, RSK2 interacted with AKTs (AKT 1-3) via the N-terminal kinase domain (NTKD) of RSK2, resulting in the phosphorylation of RSK2. The AKT-mediated phosphorylation consensus sequence, RxRxxS/T, on RSK2 NTKD (Thr115) was well conserved in different species. In particular, an in vitro kinase assay showed that NTKD deleted and Thr115Ala mutants of RSK2 abolished AKT1-mediated phosphorylation. In the physiological assay of RSK2 phosphorylation at Thr115 on cell proliferation, AKT1-mediated RSK2 phosphorylation at Thr115 played an essential role in cell proliferation. The re-introduction of RSK2-T115A to RSK2−/− MEF attenuated the EGF-induced G1/S cell cycle transition compared to RSK2-wt introducing RSK2−/− MEFs. This attenuation was observed by EGF stimulations and insulin-like growth factor-1. Overall, these results show that novel wiring of the AKT/RSKs signaling axis plays an important role in cancer cell proliferation by modulating the G1/S cell cycle transition.

Serine 897 Phosphorylation of EPHA2 Is Involved in Signaling of Oncogenic ERK1/2 Drivers in Thyroid Cancer Cells.

Background: Phosphorylation of the intracellular domain of the EPHA2 receptor tyrosine kinase (RTK) on serine 897 (S897) has been demonstrated to mediate EPHA2 oncogenic activity. Here, we show that in thyroid cancer cells harboring driver oncogenes that signal through the extracellular regulated kinase (ERK1/2) signaling pathway [rearranged RET RTK (RET/PTC), KRAS(G12R), or BRAFV600E oncogenes], EPHA2 is robustly phosphorylated on S897. EPHA2 S897 is embedded in a consensus sequence for phosphorylation by the AGC family kinases, including p90RSK (ribosomal protein S6 kinase), a direct ERK1/2 target. Methods: We show that recombinant p90RSK phosphorylates in vitro EPHA2 S897 and that treatment with chemical inhibitors targeting p90RSK or other components of the ERK1/2 pathway blunts S897 phosphorylation. Results: RNA interference-mediated knockdown combined with rescue experiments demonstrated that EPHA2 S897 phosphorylation mediates thyroid cancer cell proliferation and motility. Conclusions: These findings point to EPHA2 S897 as a crucial mediator of the oncogenic activity of the ERK1/2 signaling cascade in thyroid cancer.

T95 nucleophosmin phosphorylation as a novel mediator and marker of regulated cell death in acute kidney injury.

The function of site-specific phosphorylation of nucleophosmin (NPM), an essential Bax chaperone, in stress-induced cell death is unknown. We hypothesized that NPM threonine 95 (T95) phosphorylation both signals and promotes cell death. In resting cells, NPM exclusively resides in the nucleus and T95 is nonphosphorylated. In contrast, phosphorylated T95 NPM (pNPM T95) accumulates in the cytosol after metabolic stress, in multiple human cancer cell lines following γ-radiation, and in postischemic human kidney tissue. Based on the T95 phosphorylation consensus sequence, we hypothesized that glycogen synthase kinase-3β (GSK-3β) regulates cytosolic NPM translocation by phosphorylating T95 NPM. In a cell-free system, GSK-3β phosphorylated a synthetic NPM peptide containing T95. In vitro, bidirectional manipulation of GSK-3β activity substantially altered T95 phosphorylation, cytosolic NPM translocation, and cell survival during stress, mechanistically linking these lethal events. Furthermore, GSK-3β inhibition in vivo decreased cytosolic pNPM T95 accumulation in kidney tissue after experimental ischemia. In patients with acute kidney injury, both cytosolic NPM accumulation in proximal tubule cells and NPM-rich intratubular casts were detected in frozen renal biopsy tissue. These observations show, for the first time, that GSK-3β promotes cell death partly by phosphorylating NPM at T95, to promote cytosolic NPM accumulation. T95 NPM is also a rational therapeutic target to ameliorate ischemic renal cell injury and may be a universal injury marker in mammalian cells.

Recognition Motif of Protein Kinase C and Protein Phosphatase 2A

Protein phosphorylation plays a central role in transducing intracellular signaling and the balance between phosphorylation and dephosphorylation must be exquisitely controlled to maintain cellular homeostasis. Two central effectors of protein phosphorylation, protein kinase C (PKC) and protein phosphatase 2A (PP2A), are themselves regulated by protein phosphorylation but the mechanism of their interaction is not known. Here we identify a putative binding motif on the PKC kinase domain that is predicted to recruit PP2A via its B56 regulatory subunit. These two enzymes regulate the phosphorylation state of each other, with PP2A opposing constitutive phosphates on PKC required for structural integrity, and PKC proposed to phosphorylate B56, controlling its interaction with PP2A substrates. While many kinase consensus phosphorylation sequences are well‐characterized, emerging research into phosphatase‐recognition of substrates via short linear motifs (SLiMs) has uncovered a PP2A‐B56 SLiM comprised of L/F‐x‐x‐I/V‐x‐E. Sequence analysis of PKC reveals a potential PP2A‐B56 binding site that is conserved in conventional PKC isozymes (α, β, and γ); indeed the Glu is required for binding PP2A‐B56 and is present in a majority of the PKC family. This sequence is located on the C‐lobe of the kinase in a region involved in substrate binding. This study characterizes the effect of mutation on this motif on PP2A‐B56 binding and function, and on the phosphorylation state of PKC. Attesting to the importance of this motif, several cancer‐associated mutations have been identified in the motif, including PKCα‐E552Q and PKCβ‐E555K. Our study demonstrates that phosphatase SLiMs are able to inform new kinase/phosphatase relationships and may be a valuable predictive tool for studying signaling cascades in disease.Support or Funding InformationThis work was supported in part by the UCSD Graduate Training Program in Cellular and Molecular Pharmacology (T32 GM007752), National Science Foundation Graduate Research Fellowship Program, and NIH R35 GM122523.

A Phosphomimetic Mutation S215E in VDAC1 Interferes with Hekokinase Binding

Several charged residues in the voltage-dependent anion channel, VDAC1, have been identified to be essential for hexokinase II (HK II) binding. Phosphorylation of VDAC1 at threonine 51 by GSK3β has also been reported to detach HK II from the channel. A serine residue S215, located on the cytoplasmic loop of VDAC1, is a potential phosphorylation target based on the consensus phosphorylation sequence for GSK3β. In the present study, we investigated the effects of a phosphomimetic S215E on the functional interaction between VDAC1 and HK II. Wild-type (WT) recombinant rat VDAC1 was obtained from OriGene. S215E was generated by site-directed mutagenesis. The WT-VDAC1 or the S215E-VDAC1 protein was reconstituted in planar lipid bilayers for electrophysiological measurements. The reconstitution of the proteins in the bilayers was confirmed by the characteristic voltage-dependence of VDAC1 in response to a voltage ramp protocol from −80 to +80 mV. Following functional confirmation, a solution change prevented further insertion of additional channel proteins into the bilayer. In the absence of HK II, S215E-VDAC1 maintained the characteristic voltage-dependence as WT-VDAC1, indicating that the mutation had no significant effects on channel gating. With the addition of HK II (60 kU/ml; human recombinant HK II, Genway), WT-VDAC1 conductance decreased to 55±5% (n=8) of its initial value. This inhibition was maintained throughout a 24-min recording period. In contrast, HK II had no significant effect on the S215E-VDAC1, where conductance remained unchanged (95±7% of its initial value; n=6). These findings provide strong evidence on the critical role of charged residues in the VDAC1-HK II interactions. Furthermore, our results implicate the role of phosphorylation in modulating this interaction. Molecular dynamics simulations are ongoing to further delineate the VDAC1-HK II interactions.

Inactivated AMPK-α2 promotes the progression of diabetic brain damage by Cdk5 phosphorylation at Thr485 site

Changes in brain energy metabolism in diabetes mellitus, including increased insulin resistance and mitochondrial dysfunction, are critically involved in diabetes-related neurodegeneration, and associate with early cognitive impairment as well. The aim of this study is to detect the specific phosphorylated-Thr485- AMP-activated protein kinase (AMPK-α2), regulated by cyclin-dependent kinase 5 (Cdk5) paly the inhibitory functional role of AMPK-α2, Which is maybe the link to the accelerated diabetic brain damage progression. Here, we used GK rats, the type 2 diabetic animal model for in vivo studies and performed In vitro kinase assay, high glucose treatment, -phosphorylated mutation and protein expression in both HEK-293T and HT-22 cell lines. In vitro, the results show that murine wild-type AMPK-α2 was phosphorylated by Cdk5 at a (S/T)PX(K/H/R) phosphorylation consensus sequence, which was associated with decreased AMPK-α2 activity. Surprisingly, mutation of Thr485 to alanine in AMPK-α2 results in the abolished Cdk5 effects, demonstrating that Thr485-phosphorylation is critical to AMPK-α2 inhibition by Cdk5. In addition, these alterations in AMPK-α2-phosphorylation and -activity induced by Cdk5 is specific at Thr485. Furthermore, in GK rats, the increased phosphorylated- Thr 485 of AMPK-α2 results in the decreased AMPK-α2 activity, which is correlated with the apoptosis of neurons in hippocamps. After high glucose treatment, the decreased survival showed in AMPK-α2T485A HT-22 cells compared to AMPK-α2WT. The down-regulated of p-CREB, SNAP25, synaptophysin as well as synapsin-1were shown in both GK rats and HT-22 cell line. Meanwhile, pre-treated with either the specific Cdk5-inhibitor (roscovitine) or the antidiabetic AMPK-α2-inhibitor (metformin) could restore the alterations in neuronal protein expression. Our results suggest that Cdk5-mediated phosphorylated- Thr485 in AMPK-α2 may be involved in the pathogenesis of diabetic brain damage.

Phosphorylation of DEPDC5, a component of the GATOR1 complex, releases inhibition of mTORC1 and promotes tumor growth

The Pim and AKT serine/threonine protein kinases are implicated as drivers of cancer. Their regulation of tumor growth is closely tied to the ability of these enzymes to mainly stimulate protein synthesis by activating mTORC1 (mammalian target of rapamycin complex 1) signaling, although the exact mechanism is not completely understood. mTORC1 activity is normally suppressed by amino acid starvation through a cascade of multiple regulatory protein complexes, e.g., GATOR1, GATOR2, and KICSTOR, that reduce the activity of Rag GTPases. Bioinformatic analysis revealed that DEPDC5 (DEP domain containing protein 5), a component of GATOR1 complex, contains Pim and AKT protein kinase phosphorylation consensus sequences. DEPDC5 phosphorylation by Pim and AKT kinases was confirmed in cancer cells through the use of phospho-specific antibodies and transfection of phospho-inactive DEPDC5 mutants. Consistent with these findings, during amino acid starvation the elevated expression of Pim1 overcame the amino acid inhibitory protein cascade and activated mTORC1. In contrast, the knockout of DEPDC5 partially blocked the ability of small molecule inhibitors against Pim and AKT kinases both singly and in combination to suppress tumor growth and mTORC1 activity in vitro and in vivo. In animal experiments knocking in a glutamic acid (S1530E) in DEPDC5, a phospho mimic, in tumor cells induced a significant level of resistance to Pim and the combination of Pim and AKT inhibitors. Our results indicate a phosphorylation-dependent regulatory mechanism targeting DEPDC5 through which Pim1 and AKT act as upstream effectors of mTORC1 to facilitate proliferation and survival of cancer cells.

A Journey through the Cytoskeleton with Protein Kinase CK2.

Substrate pleiotropicity, a very acidic phosphorylation consensus sequence, and an apparent uncontrolled activity, are the main features of CK2, a Ser/Thr protein kinase that is required for a plethora of cell functions. Not surprisingly, CK2 appears to affect cytoskeletal structures and correlated functions such as cell shape, mechanical integrity, cell movement and division. This review outlines our current knowledge of how CK2 regulates cytoskeletal structures, and discusses involved pathways and molecular mechanisms.

Rational Redesign of a Functional Protein Kinase-Substrate Interaction.

Eukaryotic protein kinases typically phosphorylate substrates in the context of specific sequence motifs, contributing to specificity essential for accurate signal transmission. Protein kinases recognize their target sequences through complementary interactions within the active site cleft. As a step toward the construction of orthogonal kinase signaling systems, we have re-engineered the protein kinase Pim1 to alter its phosphorylation consensus sequence. Residues in the Pim1 catalytic domain interacting directly with a critical arginine residue in the substrate were substituted to produce a kinase mutant that instead accommodates a hydrophobic residue. We then introduced a compensating mutation into a Pim1 substrate, the pro-apoptotic protein BAD, to reconstitute phosphorylation both in vitro and in living cells. Coexpression of the redesigned kinase with its substrate in cells protected them from apoptosis. Such orthogonal kinase–substrate pairs provide tools to probe the functional consequences of specific phosphorylation events in living cells and to design synthetic signaling pathways.

Mechanisms of Yersinia YopO kinase substrate specificity

Yersinia bacteria cause a range of human diseases, including yersiniosis, Far East scarlet-like fever and the plague. Yersiniae modulate and evade host immune defences through injection of Yersinia outer proteins (Yops) into phagocytic cells. One of the Yops, YopO (also known as YpkA) obstructs phagocytosis through disrupting actin filament regulation processes - inhibiting polymerization-promoting signaling through sequestration of Rac/Rho family GTPases and by using monomeric actin as bait to recruit and phosphorylate host actin-regulating proteins. Here we set out to identify mechanisms of specificity in protein phosphorylation by YopO that would clarify its effects on cytoskeleton disruption. We report the MgADP structure of Yersinia enterocolitica YopO in complex with actin, which reveals its active site architecture. Using a proteome-wide kinase-interacting substrate screening (KISS) method, we identified that YopO phosphorylates a wide range of actin-modulating proteins and located their phosphorylation sites by mass spectrometry. Using artificial substrates we clarified YopO’s substrate length requirements and its phosphorylation consensus sequence. These findings provide fresh insight into the mechanism of the YopO kinase and demonstrate that YopO executes a specific strategy targeting actin-modulating proteins, across multiple functionalities, to compete for control of their native phospho-signaling, thus hampering the cytoskeletal processes required for macrophage phagocytosis.

Abstract PR04: Small molecule-mediated activation of Ras elicits inhibition of MAPK and PI3K signaling though pathway feedback

Abstract Oncogenic mutation or hyper-activation of Ras results in aberrant cellular signaling and is responsible for approximately 30% of all human tumors. Therefore, Ras is an outstanding candidate for therapeutic inhibition. However, the discovery of potent inhibitors has been challenging. Our laboratory has recently discovered small molecules that perturb Son of Sevenless (SOS) -catalyzed nucleotide exchange on Ras (Burns et al. PNAS 2014). Here we describe experiments conducted to determine the mechanism of compound action. SOS activator compound 4 (C4) induces signaling flux through the MAPK pathway in response to elevated Ras-GTP levels, reminiscent of epidermal growth factor signaling. We used mass spectrometry to show that in response to C4 stimulation, phospho-modifications on SOS1 increased at ERK phosphorylation consensus sequences. We hypothesize that these phospho-modifications can cause delocalization of SOS and membrane bound Ras, and we are currently using proximity-ligation to assess whether the interaction between SOS1 and Ras is affected by C4 treatment. Cross-talk between the MAPK and PI3K signaling pathways may be responsible for the inhibition of phospho-AKT Ser473 levels in response to MAPK signaling flux, or vice versa. Here we show, using MEK and PI3K inhibitor pre-treatments, that events in both pathways are independent after C4 treatment. As both SOS1 and PI3K bind to Ras at a similar site, we are currently testing the hypothesis that C4 disrupts the interaction between Ras and PI3K, whilst allowing signaling flux through the MAPK pathway. Overall, we have discovered small-molecule activators of SOS that modulate Ras-GTP levels, resulting in signaling flux through the MAPK pathway and eventual downstream inhibition of both the MAPK and PI3K pathways in cancer cells. This abstract is also being presented as Poster A15. Citation Format: Jennifer E. Howes, Denis T. Akan, Bethany M. Alicie, Alex G. Waterson, Olivia W. Rossanese, Stephen W. Fesik. Small molecule-mediated activation of Ras elicits inhibition of MAPK and PI3K signaling though pathway feedback. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr PR04.

Aurora kinase A activates the vacuolar H+-ATPase (V-ATPase) in kidney carcinoma cells.

Extracellular proton-secreting transport systems that contribute to extracellular pH include the vacuolar H(+)-ATPase (V-ATPase). This pump, which mediates ATP-driven transport of H(+) across membranes, is involved in metastasis. We previously showed (Alzamora R, Thali RF, Gong F, Smolak C, Li H, Baty CJ, Bertrand CA, Auchli Y, Brunisholz RA, Neumann D, Hallows KR, Pastor-Soler NM. J Biol Chem 285: 24676-24685, 2010) that V-ATPase A subunit phosphorylation at Ser-175 is important for PKA-induced V-ATPase activity at the membrane of kidney intercalated cells. However, Ser-175 is also located within a larger phosphorylation consensus sequence for Aurora kinases, which are known to phosphorylate proteins that contribute to the pathogenesis of metastatic carcinomas. We thus hypothesized that Aurora kinase A (AURKA), overexpressed in aggressive carcinomas, regulates the V-ATPase in human kidney carcinoma cells (Caki-2) via Ser-175 phosphorylation. We found that AURKA is abnormally expressed in Caki-2 cells, where it binds the V-ATPase A subunit in an AURKA phosphorylation-dependent manner. Treatment with the AURKA activator anacardic acid increased V-ATPase expression and activity at the plasma membrane of Caki-2 cells. In addition, AURKA phosphorylates the V-ATPase A subunit at Ser-175 in vitro and in Caki-2 cells. Immunolabeling revealed that anacardic acid induced marked membrane accumulation of the V-ATPase A subunit in transfected Caki-2 cells. However, anacardic acid failed to induce membrane accumulation of a phosphorylation-deficient Ser-175-to-Ala (S175A) A subunit mutant. Finally, S175A-expressing cells had decreased migration in a wound-healing assay compared with cells expressing wild-type or a phospho-mimetic Ser-175-to-Asp (S175D) mutant A subunit. We conclude that AURKA activates the V-ATPase in kidney carcinoma cells via phosphorylation of Ser-175 in the V-ATPase A subunit. This regulation contributes to kidney carcinoma V-ATPase-mediated extracellular acidification and cell migration.

Rapid Identification of Protein Kinase Phosphorylation Site Motifs Using Combinatorial Peptide Libraries.

Eukaryotic protein kinases phosphorylate substrates at serine, threonine, and tyrosine residues that fall within the context of short sequence motifs. Knowing the phosphorylation site motif for a protein kinase facilitates designing substrates for kinase assays and mapping phosphorylation sites in protein substrates. Here, we describe an arrayed peptide library protocol for rapidly determining kinase phosphorylation consensus sequences. This method uses a set of peptide mixtures in which each of the 20 amino acid residues is systematically substituted at nine positions surrounding a central site of phosphorylation. Peptide mixtures are arrayed in multiwell plates and analyzed by radiolabel assay with the kinase of interest. The preferred sequence is determined from the relative rate of phosphorylation of each peptide in the array. Consensus peptides based on these sequences typically serve as efficient and specific kinase substrates for high-throughput screening or incorporation into biosensors.

Identification of Phosphorylation Consensus Sequences and Endogenous Neuronal Substrates of the Psychiatric Risk Kinase TNIK.

Traf2- and Nck-interacting kinase (TNIK) is a serine/threonine kinase highly expressed in the brain and enriched in the postsynaptic density of glutamatergic synapses in the mammalian brain. Accumulating genetic evidence and functional data have implicated TNIK as a risk factor for psychiatric disorders. However, the endogenous substrates of TNIK in neurons are unknown. Here, we describe a novel selective small molecule inhibitor of the TNIK kinase family. Using this inhibitor, we report the identification of endogenous neuronal TNIK substrates by immunoprecipitation with a phosphomotif antibody followed by mass spectrometry. Phosphorylation consensus sequences were defined by phosphopeptide sequence analysis. Among the identified substrates were members of the delta-catenin family including p120-catenin, δ-catenin, and armadillo repeat gene deleted in velo-cardio-facial syndrome (ARVCF), each of which is linked to psychiatric or neurologic disorders. Using p120-catenin as a representative substrate, we show TNIK-induced p120-catenin phosphorylation in cells requires intact kinase activity and phosphorylation of TNIK at T181 and T187 in the activation loop. Addition of the small molecule TNIK inhibitor or knocking down TNIK by two shRNAs reduced endogenous p120-catenin phosphorylation in cells. Together, using a TNIK inhibitor and phosphomotif antibody, we identify endogenous substrates of TNIK in neurons, define consensus sequences for TNIK, and suggest signaling pathways by which TNIK influences synaptic development and function linked to psychiatric and neurologic disorders.

Phosphorylation of the centrosomal protein, Cep169, by Cdk1 promotes its dissociation from centrosomes in mitosis

Cep169 is a centrosomal protein conserved among vertebrates. In our previous reports, we showed that mammalian Cep169 interacts and collaborates with CDK5RAP2 to regulate microtubule (MT) dynamics and stabilization. Although Cep169 is required for MT regulation, its precise cellular function remains largely elusive. Here we show that Cep169 associates with centrosomes during interphase, but dissociates from these structures from the onset of mitosis, although CDK5RAP2 (Cep215) is continuously located at the centrosomes throughout cell cycle. Interestingly, treatment with purvalanol A, a Cdk1 inhibitor, nearly completely blocked the dissociation of Cep169 from centrosomes during mitosis. In addition, mass spectrometry analyses identified 7 phosphorylated residues of Cep169 corresponding to consensus phosphorylation sequence for Cdk1. These data suggest that the dissociation of Cep169 from centrosomes is controlled by Cdk1/Cyclin B during mitosis, and that Cep169 might regulate MT dynamics of mitotic spindle.

Phosphorylation sites in the Hook domain of CaVβ subunits differentially modulate CaV1.2 channel function

Regulation of L-type calcium current is critical for the development, function, and regulation of many cell types. CaV1.2 channels that conduct L-type calcium currents are regulated by many protein kinases, but the sites of action of these kinases remain unknown in most cases. We combined mass spectrometry (LC–MS/MS) and whole-cell patch clamp techniques in order to identify sites of phosphorylation of CaVβ subunits in vivo and test the impact of mutations of those sites on CaV1.2 channel function in vitro. Using the CaV1.1 channel purified from rabbit skeletal muscle as a substrate for phosphoproteomic analysis, we found that Ser193 and Thr205 in the HOOK domain of CaVβ1a subunits were both phosphorylated in vivo. Ser193 is located in a potential consensus sequence for casein kinase II, but it was not phosphorylated in vitro by that kinase. In contrast, Thr205 is located in a consensus sequence for cAMP-dependent phosphorylation, and it was robustly phosphorylated in vitro by PKA. These two sites are conserved in multiple CaVβ subunit isoforms, including the principal CaVβ subunit of cardiac CaV1.2 channels, CaVβ2b. In order to assess potential modulatory effects of phosphorylation at these sites separately from the effects of phosphorylation of the α11.2 subunit, we inserted phosphomimetic or phosphoinhibitory mutations in CaVβ2b and analyzed their effects on CaV1.2 channel function in transfected nonmuscle cells. The phosphomimetic mutation CaVβ2bS152E decreased peak channel currents and shifted the voltage dependence of both activation and inactivation to more positive membrane potentials. The phosphoinhibitory mutation CaVβ2bS152A had opposite effects. There were no differences in peak CaV1.2 currents or voltage dependence between the phosphomimetic mutation CaVβ2bT164D and the phosphoinhibitory mutation CaVβ2bT164A. However, calcium-dependent inactivation was significantly increased for the phosphomimetic mutation CaVβ2bT164D. This effect was subunit-specific, as the corresponding mutation in the palmitoylated isoform, CaVβ2a, had no effect. Overall, our data identify two conserved sites of phosphorylation of the Hook domain of CaVβ subunits in vivo and reveal differential modulatory effects of phosphomimetic mutations in these sites. These results reveal a new dimension of regulation of CaV1.2 channels through phosphorylation of the Hook domains of their β subunits.

Regulation of the cystic fibrosis transmembrane conductance regulator anion channel by tyrosine phosphorylation.

The cystic fibrosis transmembrane conductance regulator (CFTR) channel is activated by PKA phosphorylation of a regulatory domain that interacts dynamically with multiple CFTR domains and with other proteins. The large number of consensus sequences for phosphorylation by PKA has naturally focused most attention on regulation by this kinase. We report here that human CFTR is also phosphorylated by the tyrosine kinases p60c-Src (proto-oncogene tyrosine-protein kinase) and the proline-rich tyrosine kinase 2 (Pyk2), and they can also cause robust activation of quiescent CFTR channels. In excised patch-clamp experiments, CFTR activity during exposure to Src or Pyk2 reached ∼80% of that stimulated by PKA. Exposure to PKA after Src or Pyk2 caused a further increase to the level induced by PKA alone, implying a common limiting step. Channels became spontaneously active when v-Src or the catalytic domain of Pyk2 was coexpressed with CFTR and were further stimulated by the tyrosine phosphatase inhibitor dephostatin. Exogenous Src also activated 15SA-CFTR, a variant that lacks 15 potential PKA sites and has little response to PKA. PKA-independent activation by tyrosine phosphorylation has implications for the mechanism of regulation by the R domain and for the physiologic functions of CFTR.

Identification of AMP-activated protein kinase targets by a consensus sequence search of the proteome.

BackgroundAMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine protein kinase that is activated by cellular perturbations associated with ATP depletion or stress. While AMPK modulates the activity of a variety of targets containing a specific phosphorylation consensus sequence, the number of AMPK targets and their influence over cellular processes is currently thought to be limited.ResultsWe queried the human and the mouse proteomes for proteins containing AMPK phosphorylation consensus sequences. Integration of this database into Gaggle software facilitated the construction of probable AMPK-regulated networks based on known and predicted molecular associations. In vitro kinase assays were conducted for preliminary validation of 12 novel AMPK targets across a variety of cellular functional categories, including transcription, translation, cell migration, protein transport, and energy homeostasis. Following initial validation, pathways that include NAD synthetase 1 (NADSYN1) and protein kinase B (AKT2) were hypothesized and experimentally tested to provide a mechanistic basis for AMPK regulation of cell migration and maintenance of cellular NAD+ concentrations during catabolic processes.ConclusionsThis study delineates an approach that encompasses both in silico procedures and in vitro experiments to produce testable hypotheses for AMPK regulation of cellular processes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12918-015-0156-0) contains supplementary material, which is available to authorized users.

Endoplasmic reticulum Ca2+ content decrease by PKA-dependent hyperphosphorylation of type 1 IP3 receptor contributes to prostate cancer cell resistance to androgen deprivation

Reference treatment of advanced prostate cancer (PCa) relies on pharmacological or surgical androgen deprivation therapy. However, it is only temporarily efficient as tumor cells inevitably adapt to the low testosterone environment and become hormone-refractory (HRPCa). We observed that androgen removal in HRPCa-derived LNCaP cells causes different alterations in their Ca2+ homeostasis among which a reduction of ER Ca2+ content. We show that the decrease in [Ca2+]ER is due to a modest overexpression of type 1 IP3R and a threefold increased phosphorylation of IP3R1 on Ser-1716, a protein kinase A (PKA) consensus site, both implicated in ER Ca2+ leak. Accordingly, ER Ca2+ content was restored by siRNA-mediated down-regulation of IP3R1 or by inhibition of its phosphorylation by competition with a permeant TAT-peptide containing the Ser-1716 consensus phosphorylation sequence or by treatment with the PKA inhibitor H89. Moreover, inhibition of the IP3R1 phosphorylation by both methods sensitized the LNCaP cells to androgen deprivation-induced apoptosis. In addition, SERCA2b overexpression precluded the effect of androgen deprivation on ER Ca2+ store content and reduced resistance to androgen deprivation. Taken together, these results indicate that lowering the ER Ca2+-store content by increasing IP3R1 levels and IP3R1 phosphorylation by PKA is a protective mechanism by which HRPCa-derived cells escape cell death in the absence of androgenic stimulation.

Differential targeting of cPKC and nPKC decodes and regulates Ca²⁺ and lipid signalling.

Protein kinases C (PKCs) are ubiquitously expressed and play critical roles in a plethora of physiological and pathophysiological processes. Owing to PKCs' highly conserved phosphorylation consensus sequence, it has been difficult to distinguish the role of individual PKC isoforms. Recently, the identification of novel membrane targeting via subcellularly targeted diacylglycerol production found for novel PKCs (nPKCs), together with a characterization of their putative functions, has shed new light on the specific roles of individual PKCs in cellular processes.