Consensus Phosphorylation Sites Research Articles

Tmem16a‐induced ClCa currents are regulated via phosphotransferase activity

Ca2+‐activated Cl− currents (ICl(Ca)) represent a major excitatory mechanism in vascular smooth muscle cells (SMCs). In arterial SMCs, ICl(Ca) are down‐regulated by calmodulin‐dependent protein kinase II (CaMKII) and up‐regulated by calcineurin (CaN) and PP1. Recently, Tmem16a has emerged as a gene candidate for the native ICl(Ca) in pulmonary artery SMCs. HEK‐293 cells over‐expressing Tmem16a displayed time‐ and voltage‐dependent Cl− currents when dialyzed with 500 nM free Ca2+ that resembled the properties of ICl(Ca) from vascular SMCs. Similar to ICl(Ca) from VSMCs, Tmem16a‐induced ICl(Ca) also displayed enhanced rundown when cells were dialyzed with 5 mM ATP and this process was attenuated in the absence of ATP. Similar to ICl(Ca) in rabbit VSMCs (Ayon et al., J Biol Chem 284:32507–32521, 2009), dialysis of 30 nM okadaic acid, a nonselective PP1/PP2A inhibitor, impeded the recovery of Tmem16a‐dependent Cl− currents in the absence of ATP. Analysis of mouse Tmem16a protein sequence revealed 5 consensus sites for phosphorylation by CaMKII, which are believed to lie on protein segments facing the cytoplasm. A single point mutation of one of these putative CaMKII phosphorylation sites (Threonine 610 to Alanine) did not alter rundown of the channels. Taken together, these data suggest that much like native ICl(Ca), Tmem16a‐dependent Cl− currents also appear to be regulated by at least one phosphorylation step.

Identification of Trafficking Motifs Involved in Constitutive and Regulated Trafficking of System xc−

System xc−, a membrane transport system that exchanges glutamate for cystine, has been shown to traffic to and from the plasma membrane, enabling a rapid response to oxidative stress. Trafficking is a common form of regulation among transport proteins, and various amino acid motifs have been shown to play integral roles in the trafficking of these transporters. This study sought to identify amino acids within xCT that are involved in its trafficking. Carboxyl‐terminal truncations and point mutants of xCT were constructed in a FLAG‐tagged cDNA vector and transfected into PC12 cells. The trafficking behavior of the mutant constructs was assayed using both fluorescence microscopy and a biotinylation protocol to separate cell surface proteins from intracellular proteins. Preliminary analysis of the data suggests 1) loss of the carboxyl‐terminus results in significantly reduced cell surface expression of xCT and 2) disruption of a consensus phosphorylation site leads to greater membrane localization of xCT, providing a mechanism for rapid regulation of transporter trafficking and activity. This research was supported by the Arnold and Mable Beckman Foundation and NSF RUI # 0843564.

A Proteomic Study of Myosin II Motor Proteins during Tumor Cell Migration

Myosin II motor proteins play important roles in cell migration. Although myosin II filament assembly plays a key role in the stabilization of focal contacts at the leading edge of migrating cells, the mechanisms and signaling pathways regulating the localized assembly of lamellipodial myosin II filaments are poorly understood. We performed a proteomic analysis of myosin heavy chain (MHC) phosphorylation sites in MDA-MB 231 breast cancer cells to identify MHC phosphorylation sites that are activated during integrin engagement and lamellar extension on fibronectin. Fibronectin-activated MHC phosphorylation was identified on novel and previously recognized consensus sites for phosphorylation by protein kinase C and casein kinase II (CK-II). S1943, a CK-II consensus site, was highly phosphorylated in response to matrix engagement, and phosphoantibody staining revealed phosphorylation on myosin II assembled into leading-edge lamellae. Surprisingly, neither pharmacological reduction nor small inhibitory RNA reduction in CK-II activity reduced this stimulated S1943 phosphorylation. Our data demonstrate that S1943 phosphorylation is upregulated during lamellar protrusion, and that CK-II does not appear to be the kinase responsible for this matrix-induced phosphorylation event.

An evolutionarily acquired genotoxic response discriminates MyoD from Myf5, and differentially regulates hypaxial and epaxial myogenesis

Despite having distinct expression patterns and phenotypes in mutant mice, the myogenic regulatory factors Myf5 and MyoD have been considered to be functionally equivalent. Here, we report that these factors have a different response to DNA damage, due to the presence in MyoD and absence in Myf5 of a consensus site for Abl-mediated tyrosine phosphorylation that inhibits MyoD activity in response to DNA damage. Genotoxins failed to repress skeletal myogenesis in MyoD-null embryos; reintroduction of wild-type MyoD, but not mutant Abl phosphorylation-resistant MyoD, restored the DNA-damage-dependent inhibition of muscle differentiation. Conversely, introduction of the Abl-responsive phosphorylation motif converts Myf5 into a DNA-damage-sensitive transcription factor. Gene-dosage-dependent reduction of Abl kinase activity in MyoD-expressing cells attenuated the DNA-damage-dependent inhibition of myogenesis. The presence of a DNA-damage-responsive phosphorylation motif in vertebrate, but not in invertebrate MyoD suggests an evolved response to environmental stress, originated from basic helix-loop-helix gene duplication in vertebrate myogenesis.

The Involvement of Ser1898 of the Human L-Type Calcium Channel in Evoked Secretion

A PKA consensus phosphorylation site S1928 at the α 11.2 subunit of the rabbit cardiac L-type channel, CaV1.2, is involved in the regulation of CaV1.2 kinetics and affects catecholamine secretion. This mutation does not alter basal CaV1.2 current properties or regulation of CaV1.2 current by PKA and the beta-adrenergic receptor, but abolishes CaV1.2 phosphorylation by PKA. Here, we test the contribution of the corresponding PKA phosphorylation site of the human α 11.2 subunit S1898, to the regulation of catecholamine secretion in bovine chromaffin cells. Chromaffin cells were infected with a Semliki-Forest viral vector containing either the human wt or a mutated S1898A α 11.2 subunit. Both subunits harbor a T1036Y mutation conferring nifedipine insensitivity. Secretion evoked by depolarization in the presence of nifedipine was monitored by amperometry. Depolarization-triggered secretion in cells infected with either the wt α 11.2 or α 11.2/S1898A mutated subunit was elevated to a similar extent by forskolin. Forskolin, known to directly activate adenylyl-cyclase, increased the rate of secretion in a manner that is largely independent of the presence of S1898. Our results are consistent with the involvement of additional PKA regulatory site(s) at the C-tail of α 11.2, the pore forming subunit of CaV1.2.

Telomerase Recruitment in Saccharomyces cerevisiae Is Not Dependent on Tel1-Mediated Phosphorylation of Cdc13

In Saccharomyces cerevisiae, association between the Est1 telomerase subunit and the telomere-binding protein Cdc13 is essential for telomerase to be recruited to its site of action. A current model proposes that Tel1 binding to telomeres marks them for elongation, as the result of phosphorylation of a proposed S/TQ cluster in the telomerase recruitment domain of Cdc13. However, three observations presented here argue against one key aspect of this model. First, the pattern of Cdc13 phosphatase-sensitive isoforms is not altered by loss of Tel1 function or by mutations introduced into two conserved serines (S249 and S255) in the Cdc13 recruitment domain. Second, an interaction between Cdc13 and Est1, as monitored by a two-hybrid assay, is dependent on S255 but Tel1-independent. Finally, a derivative of Cdc13, cdc13-(S/TQ)11→(S/TA)11, in which every potential consensus phosphorylation site for Tel1 has been eliminated, confers nearly wild-type telomere length. These results are inconsistent with a model in which the Cdc13-Est1 interaction is regulated by Tel1-mediated phosphorylation of the Cdc13 telomerase recruitment domain. We propose an alternative model for the role of Tel1 in telomere homeostasis, which is based on the assumption that Tel1 performs the same molecular task at double-strand breaks (DSBs) and chromosome termini.

Palmitoylation of the S0-S1 Linker Regulates Cell Surface Expression of Voltage- and Calcium-activated Potassium (BK) Channels

S-Palmitoylation is rapidly emerging as an important post-translational mechanism to regulate ion channels. We have previously demonstrated that large conductance calcium- and voltage-activated potassium (BK) channels are palmitoylated within an alternatively spliced (STREX) insert. However, these studies also revealed that additional site(s) for palmitoylation must exist outside of the STREX insert, although the identity or the functional significance of these palmitoylated cysteine residues are unknown. Here, we demonstrate that BK channels are palmitoylated at a cluster of evolutionary conserved cysteine residues (Cys-53, Cys-54, and Cys-56) within the intracellular linker between the S0 and S1 transmembrane domains. Mutation of Cys-53, Cys-54, and Cys-56 completely abolished palmitoylation of BK channels lacking the STREX insert (ZERO variant). Palmitoylation allows the S0-S1 linker to associate with the plasma membrane but has no effect on single channel conductance or the calcium/voltage sensitivity. Rather, S0-S1 linker palmitoylation is a critical determinant of cell surface expression of BK channels, as steady state surface expression levels are reduced by ∼55% in the C53:54:56A mutant. STREX variant channels that could not be palmitoylated in the S0-S1 linker also displayed significantly reduced cell surface expression even though STREX insert palmitoylation was unaffected. Thus our work reveals the functional independence of two distinct palmitoylation-dependent membrane interaction domains within the same channel protein and demonstrates the critical role of S0-S1 linker palmitoylation in the control of BK channel cell surface expression.

Phosphorylation of CLK2 at Serine 34 and Threonine 127 by AKT Controls Cell Survival after Ionizing Radiation

AKT phosphorylates components of the intrinsic cell survival machinery and promotes survival to various stimuli. In the present study, we identified CDC-like kinase 2 (CLK2) as a new substrate of AKT activation and elucidated its role in cell survival to ionizing radiation. AKT directly binds to and phosphorylates CLK2 on serine 34 and threonine 127, in vitro and in vivo. CLK2 phosphorylation was detected in HeLa cells overexpressing active AKT. In addition, we demonstrated that ionizing radiation induces CLK2 phosphorylation via AKT activation. In contrast, the suppression of endogenous AKT expression by siRNA inhibited CLK2 phosphorylation in response to 2 gray of γ-ray or insulin. Furthermore, we examined the effect of CLK2 on the survival of irradiated CCD-18Lu cells overexpressing Myc-CLK2. CLK2 overexpression significantly increased cell growth and inhibited cell death induced by 2 gray. The role of CLK2 in cell survival to ionizing radiation was dependent on the phosphorylation of serine 34 and threonine 127. Our results suggest that AKT activation controls cell survival to ionizing radiation by phosphorylating CLK2, revealing an important regulatory mechanism required for promoting cell survival.

Regulation of Prostate Cancer Cell Survival by Protein Kinase C∈ Involves Bad Phosphorylation and Modulation of the TNFα/JNK Pathway

Protein kinase Cepsilon (PKCepsilon), a diacyglycerol- and phorbol ester-responsive serine-threonine kinase, has been implicated in mitogenic and survival control, and it is markedly overexpressed in human tumors, including in prostate cancer. Although prostate cancer cells undergo apoptosis in response to phorbol ester stimulation via PKCdelta-mediated release of death factors, the involvement of PKCepsilon in this response is not known. PKCepsilon depletion by RNAi or expression of a dominant negative kinase-dead PKCepsilon mutant potentiated the apoptotic response of PMA and sensitized LNCaP cells to the death receptor ligand TNFalpha. On the other hand, overexpression of PKCepsilon by adenoviral means protected LNCaP cells against apoptotic stimuli. Interestingly, PKCepsilon RNAi depletion significantly enhanced the release of TNFalpha in response to PMA and greatly potentiated JNK activation by this cytokine. Further mechanistic analysis revealed that PMA fails to promote phosphorylation of Bad in Ser(112) in PKCepsilon-depleted LNCaP cells, whereas PKCepsilon overexpression greatly enhanced Bad phosphorylation. This effect was independent of Akt, ERK, or p90Rsk, well established kinases for Ser(112) in Bad. Moreover, expression of a S112A-Bad mutant potentiated PMA-induced apoptosis. Finally, we found that upon activation PKCepsilon accumulated in mitochondrial fractions in LNCaP cells and that Bad was a substrate of PKCepsilon in vitro. Our results established that PKCepsilon modulates survival in prostate cancer cells via multiple pathways.

Protein kinase A catalytic subunit interacts and phosphorylates members of trans-sialidase super-family in Trypanosoma cruzi

Protein kinase A (PKA) has been suggested as a regulator of stage differentiation in Trypanosoma cruzi. Using a yeast two-hybrid system we have begun to characterize the downstream substrates of T. cruzi PKA. We identified several members of the trans-sialidase super family by this approach. Immunoprecitation demonstrated that a TcPKAc monoclonal antibody was able to pull-down proteins recognized by trans-sialidase antibodies as well as a SA85-1.1 antibody and vice versa. An in vitro phosphorylation assay demonstrated that PKA phosphorylated the recombinant protein of an active trans-sialidase. In addition, a phospho-(Ser/Thr) PKA substrate antibody detected bands on immunoblot analysis of trans-sialidase antibody precipitated proteins from parasite lysate and the media of L 6E 9 myoblasts infected with trypomastigotes as well as from a SA85-1.1 antibody precipitated proteins from parasite lysate. Immunofluorescence analysis suggested that some TcPKAc localizes to the plasma membrane surface of trypomastigotes. The identified trans-sialidases have PKA consensus phosphorylation sites located near the endoplasmic reticulum retention motif in the N-terminal. These data support that PKA phosphorylates trans-sialidase super family members in vivo.

Phosphorylation of TonEBP/OREBP by cyclin dependent kinase 5 (CDK5) mediates its NaCl‐induced nuclear translocation

High NaCl increases TonEBP/OREBP nuclear localization. We find that high NaCl increases phosphorylation of TonEBP/OREBP‐T135, located in its Auxiliary Export Domain (amino acids 132–156). Mutation of T135 to alanine decreases high NaCl‐induced nuclear localization of TonEBP/OREBP. T135 is in a consensus site for phosphorylation by protein kinase CDK5. High NaCl increases phosphorylation of CDK5‐Y15, indicating increased activity. Inhibitors of CDK5 and Kinase Dead (KD) dominant negative CDK5 reduce high NaCl‐induced nuclear localization of TonEBP/OREBP. Furthermore, CDK5 associates with TonEBP/OREBP (coimmunoprecipitation) and directly phosphorylates T135 in vitro. Over expression of CDK5 KD strongly decreases phosphorylation of T135. We do not find any regulation of TonEBP/OREBP transactivating activity by CDK5. We conclude that high NaCl stimulates CDK5 kinase activity which in turn contributes to nuclear localization of TonEBP by phosphorylating it on T135.This research was supported by the Intramural Research Program of NHLBI.

A Proteome-wide Analysis of Kinase-Substrate Network in the DNA Damage Response

The DNA damage checkpoint, consisting of an evolutionarily conserved protein kinase cascade, controls the DNA damage response in eukaryotes. Knowledge of the in vivo substrates of the checkpoint kinases is essential toward understanding their functions. Here we used quantitative mass spectrometry to identify 53 new and 34 previously known targets of Mec1/Tel1, Rad53, and Dun1 in Saccharomyces cerevisiae. Analysis of replication protein A (RPA)-associated proteins reveals extensive physical interactions between RPA-associated proteins and Mec1/Tel1-specific substrates. Among them, multiple subunits of the chromatin remodeling complexes including ISW1, ISW2, INO80, SWR1, RSC, and SWI/SNF are identified and they undergo DNA damage-induced phosphorylation by Mec1 and Tel1. Taken together, this study greatly expands the existing knowledge of the targets of DNA damage checkpoint kinases and provides insights into the role of RPA-associated chromatins in mediating Mec1 and Tel1 substrate phosphorylation in vivo.

Phosphorylation May Mediate Normal Trafficking of The Betaine/GABA Transporter (BGT1) During Hypertonic Stress in Renal Medullary Cells

BGT1 mediates uptake of the osmolyte betaine in renal medullary cells. During hypertonic (Hyp, 550 mOs) stress BGT1 is synthesized and inserted in the plasma membrane of MDCK cells. The phorbol ester PMA downregulates BGT1 by endocytic removal. To further test if PKC regulates BGT1, mutants of EGFP‐tagged BGT1 were prepared by substituting alanine at consensus sites for PKC phosphorylation and were expressed in MDCK cells. Microscopy showed that mutant T40A remained intracellular during Hyp stress, but substitution of aspartate at T40 did not interfere with the normal trafficking response. Next, native MDCK cells were incubated in Hyp growth medium for 24 h in absence (controls) or presence of PKC inhibitors (staurosporine 0.3 μM or Go6976 10 μM), or PMA (75 nM) for downregulation of PKC. In Hyp controls GABA transport was increased 10‐fold compared to isotonic groups, but the increase was only 3–4 fold when inhibitors or PMA were present (p<0.001, n=3). In contrast, amino acid transport system A was upregulated normally under all Hyp conditions. Westerns of cell lysates showed the abundance of BGT1 protein was not changed by drug treatment. However, consistent with transport data, microscopy revealed a reduction in plasma membrane BGT1 after drug treatment. Data suggest PKC‐dependent phosphorylation at T40 may be required for normal trafficking of BGT1 to the plasma membrane during Hyp stress. AHA 09GRNT2010156.

An SP/TP motif in the N‐terminal cytoplasmic domain of UT‐A3 as potential activation site of UT‐A3‐mediated urea transport

The UT‐A protein variant UT‐A3 possesses in its N‐terminal cytoplasmic domain two SP sites, which are potential phosphorylation sites by proline‐directed kinases. In rat UT‐A3, they are located at positions 15/16 and 35/36. The purpose of the experiments was to test if these sites are involved in the cAMP‐dependent activation of urea influx in UT‐A3‐expressing Xenopus oocytes. By mutating two consensus phosphorylation sites for protein kinase A (S85 and S92 of mouse UT‐A3), Smith and coworkers (Smith CP et al, AJP Cell 287:C1087, 2004) had shown that these PKA sites were not responsible for the observed cAMP dependence of UT‐A3 in oocytes. We prepared N‐terminal deletion constructs of UT‐A3 in which the deleted sequence was replaced by a start codon. Thus these constructs started with a methionine either at positions 18 (del18), 35 (del35) or 53 (del53). When expressed in Xenopus oocytes, UT‐A3‐del18 had the same urea transport activity and cAMP stimulation as wild‐type UT‐A3. However, UT‐A3‐del35 and UT‐A3‐del53, while exhibiting the same unstimulated urea transport activity, were not stimulated by cAMP treatment. To test the hypothesis that the SP35 site is involved in the activation of UT‐A3, we mutated the S15 and S35 either alone or together. As expected, UT‐A3‐S15A behaved like wild‐type UT‐A3 whereas S35A and S15A/S35A were no longer stimulated by cAMP treatment. These data suggest that SP35 is responsible for the activation of UT‐A3 in oocytes. They are also consistent with the findings of the proteomic study of Hoffert et al. (PNAS 103:7159, 2006) who found that S35 was phosphorylated in response to vasopressin in the renal inner medulla. This research was supported by NIH grant DK41707 and the Emory University Research Council

Sequence Alignment Reveals Possible MAPK Docking Motifs on HIV Proteins

Over the course of HIV infection, virus replication is facilitated by the phosphorylation of HIV proteins by human ERK1 and ERK2 mitogen-activated protein kinases (MAPKs). MAPKs are known to phosphorylate their substrates by first binding with them at a docking site. Docking site interactions could be viable drug targets because the sequences guiding them are more specific than phosphorylation consensus sites. In this study we use multiple bioinformatics tools to discover candidate MAPK docking site motifs on HIV proteins known to be phosphorylated by MAPKs, and we discuss the possibility of targeting docking sites with drugs. Using sequence alignments of HIV proteins of different subtypes, we show that MAPK docking patterns previously described for human proteins appear on the HIV matrix, Tat, and Vif proteins in a strain dependent manner, but are absent from HIV Rev and appear on all HIV Nef strains. We revise the regular expressions of previously annotated MAPK docking patterns in order to provide a subtype independent motif that annotates all HIV proteins. One revision is based on a documented human variant of one of the substrate docking motifs, and the other reduces the number of required basic amino acids in the standard docking motifs from two to one. The proposed patterns are shown to be consistent with in silico docking between ERK1 and the HIV matrix protein. The motif usage on HIV proteins is sufficiently different from human proteins in amino acid sequence similarity to allow for HIV specific targeting using small-molecule drugs.

Contribution of Phosphorylation Residues in the A4 Subunit to the α4β2 Neuronal Nachr Function and Expression

The α4β2 nicotinic acetylcholine receptor (nAChR) is the most highly expressed subtype in the brain and plays an important role in nicotine addiction. Nicotine exerts its action on the brain, mainly on this subtype. Chronic nicotine exposure up-regulates the α4β2 nAChR, increasing the number of receptors in the CNS. The working hypothesis of this study is that the nicotine-induced up-regulation of α4β2 nAChRs may involve subunit phosphorylation. There are thirteen sites in the cytoplasmic loop of the α4 subunit, known to be consensus sites for various protein kinases. Eleven of these sites were mutated, to aspartic acid, to mimic the effect of subunit phosphorylation and to alanine to abolish a potential phosphorylation. To asses receptor function two-electrode voltage clamp was done on the mutated receptors expressed in Xenopus laevis oocytes. Binding assays using [125I]-Epibatidine was used to verify the expression of the mutated receptors. Several positions resulted in an apparent “on/off” switch for constitutive function; the “on” switch being the alanine mutation and the “off” switch being the aspartic acid substitution. For example, two alanine mutants, which are PKC phosphorylation residues, resulted in functional receptors with a macroscopic current and expression similar or greater than that of the wild-type α4β2 nAChR. On the other hand the aspartic acid mutants displayed expression levels similar to wild-type but exhibited a dramatic decrease in macroscopic current. These results suggest that these consensus phosphorylation sites can regulate the α4β2 nAChR functional responses without affecting expression. On the other hand, the increase in the EC50 for ACh displayed by several mutations is consistent with the functional parameters of nicotine-induced up-regulated α4β2 nAChRs. This suggests that phosphorylation of the α4 subunit may regulate the α4β2 nAChR function and expression.

Protein kinase C and rho activated coiled coil protein kinase 2 (ROCK2) modulate Alzheimer's APP metabolism and phosphorylation of the Vps10-domain protein, SorL1

BackgroundGeneration of the amyloid β (Aβ) peptide of Alzheimer's disease (AD) is differentially regulated through the intracellular trafficking of the amyloid β precursor protein (APP) within the secretory and endocytic pathways. Protein kinase C (PKC) and rho-activated coiled-coil kinases (ROCKs) are two "third messenger" signaling molecules that control the relative utilization of these two pathways. Several members of the Vps family of receptors (Vps35, SorL1, SorCS1) play important roles in post-trans-Golgi network (TGN) sorting and generation of APP derivatives, including Aβ at the TGN, endosome and the plasma membrane. We now report that Vps10-domain proteins are candidate substrates for PKC and/or ROCK2 and act as phospho-state-sensitive physiological effectors for post-TGN sorting of APP and its derivatives.ResultsAnalysis of the SorL1 cytoplasmic tail revealed multiple consensus sites for phosphorylation by protein kinases. SorL1 was subsequently identified as a phosphoprotein, based on sensitivity of its electrophoretic migration pattern to calf intestine alkaline phosphatase and on its reaction with anti-phospho-serine antibodies. Activation of PKC resulted in increased shedding of the ectodomains of both APP and SorL1, and this was paralleled by an apparent increase in the level of the phosphorylated form of SorL1. ROCK2, the neuronal isoform of another protein kinase, was found to form complexes with SorL1, and both ROCK2 inhibition and ROCK2 knockdown enhanced generation of both soluble APP and Aβ.ConclusionThese results highlight the potential importance of SorL1 in elucidating phospho-state sensitive mechanisms in the regulation of metabolism of APP and Aβ by PKC and ROCK2.

Characterization of an Activating Transcription Factor 4 Gene Containing a Consensus Phosphorylation Site for PKA in the Gonads ofXenopusEmbryos

Activating transcription factor / cyclic-AMP response element-binding protein (ATF/CREB) has been implicated as a key regulator in the transcriptional control of many genes. In this study, we isolated and characterized a full-length cDNA that encodes a CRE-binding protein 2 (CREB2) called ATF4 in Xenopus embryos. Like other CREB 2 transcription factors, the 342-amino acid ATF4 protein contains a carboxyl terminal leucine-zipper motif, an adjacent basic domain, and an amino terminal leucine-zipper motif. Unlike other CREB2 (ATF4) proteins, the ATF4 isolated from the gonads of Xenopus embryos contains a consensus phosphorylation site for protein kinase A (PKA). In a gel shift analysis, ATF4 bound to a CLS sequence in the promoter of Xenopus aromatase.

Molecular Mechanisms for Phosphorylation Driven Dissociation of Rb-E2F Complexes

The Retinoblastoma Protein (Rb) functions as a negative regulator of cell growth in part by physically sequestering and repressing the transactivation activity of E2F. It is well established that phosphorylation of Rb by Cyclin Dependent Kinases disrupts binding between Rb and E2F, however it is unknown which of the 15 CDK consensus phosphorylation sites on Rb are required to disrupt the interaction between the pocket domain of Rb and the transactivation domain of E2F (E2FTD). In this work, we use calorimetric assays to reveal that phosphorylation at S608/S612 and T356/T373 are together sufficient to reduce the affinity of E2FTD for Rb pocket 250-fold, the same as fully-phosphorylated Rb. Nuclear Magnetic Resonance is used to identify the phosphorylation-dependent conformational changes that directly inhibit E2FTD binding. Specifically, we have found that phosphorylation of S608/S612 promotes intramolecular binding between the flexible pocket linker and the pocket domain of Rb, while phosphorylation at T356/T373 enhances binding between the N-terminal domain and pocket domain of Rb. Taken together, our results reveal two novel mechanisms for how phosphorylation of Rb modulates binding between E2FTD and Rb pocket, and we describe for the first time a function for the N-terminal domain in the inactivation of Rb.

Rho kinase–dependent activation of SOX9 in chondrocytes

The transcription factor SOX9 directly regulates the expression of the major proteoglycans and collagens comprising the cartilage extracellular matrix. The DNA binding activity and cellular localization of SOX9 is controlled through posttranslational modifications, including phosphorylation. The activity of Rho kinase (ROCK) has profound effects on the actin cytoskeleton, and these effects are instrumental in determining the phenotype and differentiation of chondrocytes. However, the mechanisms linking ROCK to altered chondrocyte gene expression remain unknown. The purpose of the present study was to test for a direct interaction between ROCK and SOX9. Human SW1353 chondrosarcoma cells were transfected with constructs coding for RhoA, ROCK, Lim kinase, and SOX9. The interaction between ROCK and SOX9 was tested on purified proteins, and was verified within a cellular context using induced overexpression and activation of the Rho pathway. The effects of SOX9 transcriptional activation were quantified with a luciferase reporter plasmid containing SOX9 binding sites from the COL2A1 enhancer element. SOX9 was found to contain a consensus phosphorylation site for ROCK. In vitro, ROCK directly phosphorylated SOX9 at Ser(181), and the overexpression of ROCK or the activation of the RhoA pathway in SW1353 chondrosarcoma cells increased SOX9(Ser181) phosphorylation. ROCK caused a dose-dependent increase in the transcription of a SOX9-luciferase reporter construct, and increased phosphorylation and nuclear accumulation of SOX9 protein in response to transforming growth factor beta treatment and mechanical compression. These results demonstrate a new interaction that directly links ROCK to increased cartilage matrix production via activation of SOX9 in response to mechanical and growth factor stimulation.