Individual Phosphorylation Sites Research Articles

CDK4 Phosphorylation Regulates Smad3 Activity in Cyclin D1 Overexpressing Breast Cancer Cells.

Abstract Introduction: Overexpression of cyclin D1 is associated with poor prognosis in breast cancer. Cyclin D1 regulates the noncanonical phosphorylation of Smad3, a member of the TGFΒ signaling cascade, through CDK4 and inhibits Smad3 activity in normal mouse fibroblasts and epithelial cells. We hypothesize that overexpression of cyclin D1 exerts tumorigenic effects in breast cancer cells through a CDK4 mediated inhibition of Smad3 cell cycle control.Methods: To examine the impact of cyclin D1 overexpression on Smad3 function, constructs containing empty vector (V), WT Smad3 (WT), and Smad3 with single or multiple CDK phosphorylation site mutations were cotransfected with a Smad3 reporter into cyclin D1 overexpressing MCF-7 and T47D cells. Smad3 containing mutant CDK sites is resistant to inhibitory CyclinD1/CDK4 phosphorylation. Study cells were also transfected with V, WT or mutant Smad3 and treated with a CDK4 inhibitor. Smad3 function was examined by luciferase reporter assays and cell cycle analysis. Levels of mRNA for cell cycle regulators cyclin dependent kinase inhibitor (cdki) p15 and c-myc were measured by qRT-PCR.Results: As expected, higher amounts of c-myc mRNA and lower amounts of cdki p15 mRNA were observed in parental, vector control and cyclin D1 overexpressing cells. While subsequent transfection with WT Smad3 resulted in a decrease in c-myc and an increase in p15 mRNA levels, the greatest decrease in c-myc and increase in p15 mRNA was found after transfection with CDK mutated Smad3, most prominently in the cyclin D1 overexpressing cells. Transfection of all individual Smad3 CDK phosphorylation site mutations resulted in an increase in Smad3 reporter activity as compared to transfection with WT Smad3. Transfection of Smad3 constructs with 4 and 5 CDK sites mutated resulted in highest overall Smad3 reporter activity, compared to WT Smad3, in the study cells. A dose dependent increase in Smad3 reporter activity was shown when MCF-7 and T47D cells were transfected with WT Smad3 and treated with increasing concentrations of a specific CDK4 inhibitor. Comparatively higher Smad3 reporter activity was found when the study cells were transfected with individual Smad3 mutant constructs and treated with the CDK4 inhibitor. Interestingly, transfection with the Smad3 construct with all CDK sites mutated and treatment with the CDK4 inhibitor resulted in inhibition of Smad3 reporter activity in the study cells. Lastly, cell cycle analysis revealed that MCF-7 and T47D cells treated with the CDK4 inhibitor showed an increase in the G1 cell cycle population.Conclusions: Mutation of CDK phosphorylation sites in the Smad3 construct or direct inhibition of CDK4 appear to facilitate release of cyclin D1/CDK4 mediated inhibition of Smad3 in breast cancer cells. To this end, restoration of Smad3 activity resulted in increasing p15 and decreasing c-myc mRNA levels in the study cells. However, mutation of all Smad3 CDK phosphorylation sites and treatment with the CDK4 inhibitor resulted in a decrease in Smad3 reporter activity, indicating that some CDK activity is necessary for Smad3 function. By helping to restore cell cycle control, inhibition of CDK4 activity may have a role in the treatment of primary and metastatic breast cancers overexpressing cyclin D. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2152.

Cdc2-mediated Phosphorylation of CLIP-170 Is Essential for Its Inhibition of Centrosome Reduplication

CLIP-170, the founding member of microtubule "plus ends tracking" proteins, is involved in many critical microtubule-related functions, including recruitment of dynactin to the microtubule plus ends and formation of kinetochore-microtubule attachments during metaphase. Although it has been reported that CLIP-170 is a phosphoprotein, neither have individual phosphorylation sites been identified nor have the associated kinases been extensively studied. Herein, we identify Cdc2 as a kinase that phosphorylates CLIP-170. We show that Cdc2 interacts with CLIP-170 mediating its phosphorylation on Thr(287) in vivo. Significantly, expression of CLIP-170 with a threonine 287 to alanine substitution (T287A) results in its mislocalization, accumulation of Plk1 and cyclin B, and block of the G2/M transition. Finally, we found that depletion of CLIP-170 leads to centrosome reduplication and that Cdc2 phosphorylation of CLIP-170 is required for the process. These results demonstrate that Cdc2-mediated phosphorylation of CLIP-170 is essential for the normal function of this protein during cell cycle progression.

Determination of new phosphorylation sites within natriuretic peptide receptors using mass spectrometric methods

Background While it has long been know that phosphorylation of the kinase homology domain is a key regulatory mechanism of natriuretic peptide receptors, no biochemical proof for individual phosphorylation sites has been reported. Here, we describe biochemical verification of previously identified phosphorylation sites in both natriuretic peptide receptor A (NPR-A) and natriuretic peptide receptor B (NPR-B) as well as the identification of novel phosphorylation sites in both receptors.

Host Cell Interactome of Tyrosine-Phosphorylated Bacterial Proteins

Selective interactions between tyrosine-phosphorylated proteins and their cognate, SH2-domain containing ligands play key roles in mammalian signal transduction. Several bacterial pathogens use secretion systems to inject tyrosine kinase substrates into host cells. Upon phosphorylation, these effector proteins recruit cellular binding partners to manipulate host cell functions. So far, only a few interaction partners have been identified. Here we report the results of a proteomic screen to systematically identify binding partners of all known tyrosine-phosphorylated bacterial effectors by high-resolution mass spectrometry. We identified 39 host interactions, all mediated by SH2 domains, including four of the five already known interaction partners. Individual phosphorylation sites recruited a surprisingly high number of cellular interaction partners suggesting that individual phosphorylation sites can interfere with multiple cellular signaling pathways. Collectively, our results indicate that tyrosine-phosphorylation sites of bacterial effector proteins have evolved as versatile interaction modules that can recruit a rich repertoire of cellular SH2 domains.

Contractile Response to Endothelin in Myocytes Expressing Troponin I Ser43/45 Substitution Mutants

Activation of protein kinase C (PKC) by the neurohormone, endothelin increases peak shortening and accelerates relaxation in adult rat cardiac myocytes. Endothelin-activated PKC also phosphorylates cardiac troponin I (cTnI), and our laboratory previously demonstrated a temporal role for the cTnIThr144 and cTnISer23/24 phosphorylation sites in the accelerated relaxation response to endothelin. The goal of the present study is to determine whether cTnISer43/45, a third potential target for PKC-mediated phosphorylation, plays a significant role in the contractile response to endothelin. Contractile function in adult rat cardiac myocytes expressing either cardiac troponin I with Ser43/45Ala (cTnIS43/45A) or Ser43/45Asp (cTnIS43/45D) substitutions was measured 4 days after gene transfer. Western analysis demonstrated 85-90% replacement of endogenous cTnI with cTnIFLAG, cTnIS43/45AFLAG, or cTnIS43/45DFLAG by 4 days post-gene transfer. The amplitude of sarcomere shortening increased 13.2+4.6% and the return velocity, a measure of relaxation rate increased 14.4+6.3% (n=14) in response to 10 nM endothelin over 15 min. In myocytes expressing cTnIS43/45A or cTnIS43/45D, the relaxation response to endothelin was similar to myocytes expressing cTnI or cTnIFLAG over the same time interval. The substitution mutant, cTnISer43/45Ala/Thr144Pro (cTnIA2P) was then studied to further investigate the role of individual cTnI phosphorylation sites in the endothelin response. Gene transfer of cTnIA2P produced significant cTnI replacement in preliminary studies of adult myocytes. However, in contrast to cTnIS43/45A or cTnIS43/45D, the accelerated relaxation response to 15 min of 10 nM endothelin was reduced in myocytes expressing cTnIA2P compared to cTnI-expressing myocytes (-2.3+ 4.7%, n=3). These preliminary results suggest Thr144, but not Ser43/45 plays a role in accelerating relaxation rate during the initial response to PKC activation by endothelin.

Store-operated Ca2+ Entry Is Suppressed During Mitosis Due To Phosphorylation Of The Endoplasmic Reticulum Ca2+ Sensor Stim1

When ER Ca2+ stores are depleted due to physiological Ca2+ release or pharmacological perturbation, Ca2+ influx via plasma membrane Ca2+ channels is activated by a process known as store-operated Ca2+ entry (SOCE). The current associated with SOCE is Ca2+ release-activated Ca2+ current (Icrac). SOCE involves Orai Ca2+ influx channels and STIM ER Ca2+ sensors. When ER Ca2+ stores are full, STIM1 is localized throughout the ER membrane; however, ER Ca2+ store depletion induces rearrangement of STIM1 into punctate structures near the plasma membrane where it activates Orai channels. Interestingly, mitosis is the only known physiological situation in which Ca2+ store depletion is dissociated from SOCE or Icrac activation. Identification of the molecular components of the SOCE signaling pathway has facilitated analysis of the mechanism underlying mitotic SOCE suppression. We found that in mitotic HeLa cells, an enhanced yellow fluorescent protein-tagged STIM1 (eYFP-STIM1) did not rearrange into puncta in response to Ca2+ store depletion and accordingly, SOCE was not activated. We hypothesized that mitosis-specific phosphorylation of STIM1 may underlie the block of STIM1 rearrangement and SOCE suppression. To this end, the phospho-specific MPM-2 antibody recognized eYFP-STIM1 immunoprecipitated from mitotic but not interphase cells. MPM-2 recognizes phosphorylated serine or threonine followed by proline, and human STIM1 contains 10 instances of S/T-P, all located in the cytoplasmic, C-terminus. STIM1 truncation mutants indicate that at least 2 sites within the C-terminus account for the mitosis-specific phosphorylation. Individual phosphorylation site mutants are being created to identify specific phosphorylated residues and to determine the functional consequences of phosphorylation during mitosis. Suppression of SOCE during mitosis may be an important signaling event, because mitotic processes such as chromosome separation and cytokinesis are exquisitely sensitive to small changes in cytoplasmic Ca2+.

PDGF receptor activation induces p120-catenin phosphorylation at serine 879 via a PKCα-dependent pathway

p120-catenin (p120) is required for cadherin stability and is thought to have a central role in modulating cell–cell adhesion. Several lines of evidence suggest that S/T phosphorylation may regulate p120 activity, but the upstream kinases involved have not been established, nor has a discreet measurable function been assigned to an individual site. To approach these issues, we have generated p120 phospho-specific monoclonal antibodies to several individual phosphorylation sites and are using them to pinpoint upstream kinases and signaling pathways that control p120 activity. Protein Kinase C (PKC) has been implicated as a signaling intermediate in several cadherin-associated cellular activities. Signaling events that activate PKC induce rapid phosphorylation at p120 Serine 879 (S879), suggesting that p120 activity is regulated, in part, by one or more PKC isoforms. Here, we find that physiologic activation of a G-protein coupled receptor (i.e., endothelin receptor), as well as several Receptor Tyrosine Kinases, induce rapid and robust p120 phosphorylation at S879, suggesting that these pathways crosstalk to cadherin complexes via p120. Using Va2 cells and PDGF stimulation, we show for the first time that PDGFR-mediated phosphorylation at this site is dependent on PKCα, a conventional PKC isoform implicated previously in disruption of adherens junctions.

P2-142: Decreased protein phosphatase 2A in Down syndrome brain: A cause of neurofibrillary degeneration

Individuals with Down syndrome (DS) develop neurofibrillary tangles, a characteristic brain lesion of Alzheimer's disease (AD), when they reach the fourth decade of life. Neurofibrillary tangles are composed of highly aggregated and phosphorylated form of microtubule associated protein tau. In AD, hyperphosphorylation of tau partially results from down-regulation of protein phosphatase (PP) 2A, a major brain tau phosphatase. The abnormal hyperphosphorylation of tau in DS had not yet been characterized, and its causes were not understood. The levels and site-specific phosphorylation of tau as well as the levels of several brain PPs in the temporal cortices of 6 DS (58.8 ± 3.8 years old) and 6 controls (68.0 ± 9.5 years old) were determined by using quantitative Western blot analysis. Linear correlation between the levels of PP2A catalytic subunit and the level of tau or tau phosphorylation at individual phosphorylation sites was analyzed by using Microsoft Excel 2003. We found that the level of the catalytic subunit of PP2A, but not of PP1, PP2B or PP5, was dramatically decreased. The decrease of PP2A level correlated negatively to tau level and tau phosphorylation at several abnormal hyperphosphorylation sites, including Ser199, Thr205, Thr212, Ser262, Ser396 and Ser422. These findings indicate that the down-regulation of PP2A might be involved in the abnormal hyperphosphorylation and accumulation of tau and thus neurofibrillary degeneration in DS.

Eliminating phosphorylation sites of the parathyroid hormone receptor type 1 differentially affects stimulation of phospholipase C and receptor internalization

The parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor (PTH1R) belongs to family B of seven-transmembrane-spanning receptors and is activated by PTH and PTHrP. Upon PTH stimulation, the rat PTH1R becomes phosphorylated at seven serine residues. Elimination of all PTH1R phosphorylation sites results in prolonged cAMP accumulation and impaired internalization in stably transfected LLC-PK1 cells. The present study explores the role of individual PTH1R phosphorylation sites in PTH1R signaling through phospholipase C, agonist-dependent receptor internalization, and regulation by G protein-coupled receptor kinases. By means of transiently transfected COS-7 cells, we demonstrate that the phosphorylation-deficient (pd) PTH1R confers dramatically enhanced coupling to G(q/11) proteins upon PTH stimulation predominantly caused by elimination of Ser(491/492/493), Ser(501), or Ser(504). Reportedly, impaired internalization of the pd PTH1R, however, is not dependent on a specific phosphorylation site. In addition, we show that G protein-coupled receptor kinase 2 interferes with pd PTH1R signaling to G(q/11) proteins at least partially by direct binding to G(q/11) proteins.

Temporal Analysis of Sucrose-induced Phosphorylation Changes in Plasma Membrane Proteins of Arabidopsis

Sucrose is the main product of photosynthesis and the most common transport form of carbon in plants. In addition, sucrose is a compound that serves as a signal affecting metabolic flux and development. Here we provide first results of externally induced phosphorylation changes of plasma membrane proteins in Arabidopsis. In an unbiased approach, seedlings were grown in liquid medium with sucrose and then depleted of carbon before sucrose was resupplied. Plasma membranes were purified, and phosphopeptides were enriched and subsequently analyzed quantitatively by mass spectrometry. In total, 67 phosphopeptides were identified, most of which were quantified over five time points of sucrose resupply. Among the identified phosphorylation sites, the well described phosphorylation site at the C terminus of plasma membrane H(+)-ATPases showed a relative increase in phosphorylation level in response to sucrose. This corresponded to a significant increase of proton pumping activity of plasma membrane vesicles from sucrose-supplied seedlings. A new phosphorylation site was identified in the plasma membrane H(+)-ATPase AHA1 and/or AHA2. This phosphorylation site was shown to be crucial for ATPase activity and overrode regulation via the well known C-terminal phosphorylation site. Novel phosphorylation sites were identified for both receptor kinases and cytosolic kinases that showed rapid increases in relative intensities after short times of sucrose treatment. Seven response classes were identified including non-responsive, rapid increase (within 3 min), slow increase, and rapid decrease. Relative quantification of phosphorylation changes by phosphoproteomics provides a means for identification of fast responses to external stimuli in plants as a basis for further functional characterization.

Ps in the (Channel) Pod Are Not Alike …

Bidirectional Activity-Dependent Regulation of Neuronal Ion Channel Phosphorylation. Misonou H, Menegola M, Mohapatra DP, Guy LK, Park KS, Trimmer JS. J Neurosci 2006;26(52):13505–13514. Activity-dependent dephosphorylation of neuronal Kv2.1 channels yields hyperpolarizing shifts in their voltage-dependent activation and homoeostatic suppression of neuronal excitability. We recently identified 16 phosphorylation sites that modulate Kv2.1 function. Here, we show that in mammalian neurons, compared with other regulated sites, such as serine (S)563, phosphorylation at S603 is supersensitive to calcineurin-mediated dephosphorylation in response to kainate-induced seizures in vivo, and brief glutamate stimulation of cultured hippocampal neurons. In vitro calcineurin digestion shows that supersensitivity of S603 dephosphorylation is an inherent property of Kv2.1. Conversely, suppression of neuronal activity by anesthetic in vivo causes hyperphosphorylation at S603 but not S563. Distinct regulation of individual phosphorylation sites allows for graded and bidirectional homeostatic regulation of Kv2.1 function. S603 phosphorylation represents a sensitive bidirectional biosensor of neuronal activity.

Phosphorylation of synapsin domain A is required for post-tetanic potentiation

Post-tetanic potentiation (PTP) is a form of homosynaptic plasticity important for information processing and short-term memory in the nervous system. The synapsins, a family of synaptic vesicle (SV)-associated phosphoproteins, have been implicated in PTP. Although several synapsin functions are known to be regulated by phosphorylation by multiple protein kinases, the role of individual phosphorylation sites in synaptic plasticity is poorly understood. All the synapsins share a phosphorylation site in the N-terminal domain A (site 1) that regulates neurite elongation and SV mobilization. Here, we have examined the role of phosphorylation of synapsin domain A in PTP and other forms of short-term synaptic enhancement (STE) at synapses between cultured Helix pomatia neurons. To this aim, we cloned H. pomatia synapsin (helSyn) and overexpressed GFP-tagged wild-type helSyn or site-1-mutant helSyn mutated in the presynaptic compartment of C1-B2 synapses. We found that PTP at these synapses depends both on Ca2+/calmodulin-dependent and cAMP-dependent protein kinases, and that overexpression of the non-phosphorylatable helSyn mutant, but not wild-type helSyn, specifically impairs PTP, while not altering facilitation and augmentation. Our findings show that phosphorylation of site 1 has a prominent role in the expression of PTP, thus defining a novel role for phosphorylation of synapsin domain A in short-term homosynaptic plasticity.

Regulation of PTEN Activity by Its Carboxyl-terminal Autoinhibitory Domain

The regulation of PTEN intrinsic biochemical properties has not been fully elucidated. In this report, we investigated the role of the PTEN carboxyl-terminal tail domain in regulating its membrane targeting and catalytic functions. Characterization of a panel of PTEN phosphorylation site mutants revealed that mutating Ser-385 to alanine (S385A) promoted membrane localization in vivo and phosphatase activity in vitro. Furthermore, S385A mutation was associated with a substantial reduction in the phosphorylation of the Ser-380/Thr-382/Thr-383 cluster. Therefore, Ser-385 could prime additional dephosphorylation events to regulate PTEN catalytic activity. Moreover, substituting Ser-380/Thr-382/Thr-383 to phosphomimic residues reversed the phosphatase activity of the S385A mutation. Next, we further defined the underlying mechanisms responsible for the COOH-terminal tail region in modulating PTEN biological activity. We have identified an interaction between the 71-amino acid carboxyl-terminal tail region and the CBRIII motif of the C2 domain, which has been implicated in membrane binding. In addition, a synthetic phosphomimic peptide encompassing the phosphorylation site cluster between amino acids 368 and 390 within the tail region mediated the suppression of PTEN catalytic activity in vitro. This same peptide when expressed in cultured cells also impeded PTEN membrane localization and enhanced phospho-Akt levels. Thus, our data suggest that the COOH-terminal tail can act as an autoinhibitory domain to control both PTEN membrane recruitment and phosphatase activity.

Coregulated Ataxia Telangiectasia-mutated and Casein Kinase Sites Modulate cAMP-response Element-binding Protein-Coactivator Interactions in Response to DNA Damage

The cyclic AMP-response element-binding protein (CREB) is a bZIP family transcription factor implicated as an oncoprotein and neuron survival factor. CREB is activated in response to cellular stimuli, including cAMP and Ca(2+), via phosphorylation of Ser-133, which promotes interaction between the kinase-inducible domain (KID) of CREB and the KID-interacting domain of CREB-binding protein (CBP). We previously demonstrated that the interaction between CREB and CBP is inhibited by DNA-damaging stimuli through a mechanism whereby CREB is phosphorylated by the ataxia telangiectasia-mutated (ATM) protein kinase. We now show that the ATM phosphorylation sites in CREB are functionally intertwined with a cluster of coregulated casein kinase (CK) sites. We demonstrate that DNA damage-induced phosphorylation of CREB occurs in three steps. The initial event in the CREB phosphorylation cascade is the phosphorylation of Ser-111, which is carried out by CK1 and CK2 under basal conditions and by ATM in response to ionizing radiation. The phosphorylation of Ser-111 triggers the CK2-dependent phosphorylation of Ser-108 and the CK1-dependent phosphorylation of Ser-114 and Ser-117. The phosphorylation of Ser-114 and Ser-117 by CK1 then renders CREB permissive for ATM-dependent phosphorylation on Ser-121. Mutation of Ser-121 alone abrogates ionizing radiation-dependent repression of CREB-CBP complexes, which can be recapitulated using a CK1 inhibitor. Our findings outline a complex mechanism of CREB phosphorylation in which coregulated ATM and CK sites control CREB transactivation potential by modulating its CBP-binding affinity. The coregulated ATM and CK sites identified in CREB may constitute a signaling motif that is common to other DNA damage-regulated substrates.

Bidirectional Activity-Dependent Regulation of Neuronal Ion Channel Phosphorylation

Activity-dependent dephosphorylation of neuronal Kv2.1 channels yields hyperpolarizing shifts in their voltage-dependent activation and homoeostatic suppression of neuronal excitability. We recently identified 16 phosphorylation sites that modulate Kv2.1 function. Here, we show that in mammalian neurons, compared with other regulated sites, such as serine (S)563, phosphorylation at S603 is supersensitive to calcineurin-mediated dephosphorylation in response to kainate-induced seizures in vivo, and brief glutamate stimulation of cultured hippocampal neurons. In vitro calcineurin digestion shows that supersensitivity of S603 dephosphorylation is an inherent property of Kv2.1. Conversely, suppression of neuronal activity by anesthetic in vivo causes hyperphosphorylation at S603 but not S563. Distinct regulation of individual phosphorylation sites allows for graded and bidirectional homeostatic regulation of Kv2.1 function. S603 phosphorylation represents a sensitive bidirectional biosensor of neuronal activity.

Highlights From The Literature

Highlights From The Literature

Microtubule Binding and Clustering of Human Tau-4R and Tau-P301L Proteins Isolated from Yeast Deficient in Orthologues of Glycogen Synthase Kinase-3β or cdk5

Phosphorylation of Tau protein and binding to microtubules is complex in neurons and was therefore studied in the less complicated model of humanized yeast. Human Tau was readily phosphorylated at pathological epitopes, but in opposite directions regulated by kinases Mds1 and Pho85, orthologues of glycogen synthase kinase-3beta and cdk5, respectively (1). We isolated recombinant Tau-4R and mutant Tau-P301L from wild type, Delta mds1 and Delta pho85 yeast strains and measured binding to Taxol-stabilized mammalian microtubules in relation to their phosphorylation patterns. Tau-4R isolated from yeast lacking mds1 was less phosphorylated and bound more to microtubules than Tau-4R isolated from wild type yeast. Paradoxically, phosphorylation of Tau-4R isolated from kinase Pho85-deficient yeast was dramatically increased resulting in very poor binding to microtubules. Dephosphorylation promoted binding to microtubules to uniform high levels, excluding other modifications. Isolated hyperphosphorylated, conformationally altered Tau-4R completely failed to bind microtubules. In parallel to Tau-4R, we expressed, isolated, and analyzed mutant Tau-P301L. Total dephosphorylated Tau-4R and Tau-P301L bound to microtubules very similarly. Surprisingly, Tau-P301L isolated from all yeast strains bound to microtubules more extensively than Tau-4R. Atomic force microscopy demonstrated, however, that the high apparent binding of Tau-P301L was due to aggregation on the microtubules, causing their deformation and bundling. Our data explain the pathological presence of granular Tau aggregates in neuronal processes in tauopathies.

Individual Cas Phosphorylation Sites Are Dispensable for Processive Phosphorylation by Src and Anchorage-independent Cell Growth

Cas is a multidomain signaling protein that resides in focal adhesions. Cas possesses a large central substrate domain containing 15 repeats of the sequence YXXP, which are phosphorylated by Src. The phosphorylation sites are essential for the roles of Cas in cell migration and in regulation of the actin cytoskeleton. We showed previously that Src catalyzes the multisite phosphorylation of Cas via a processive mechanism. In this study, we created mutant forms of Cas to identify the determinants for processive phosphorylation. Mutants containing single or multiple YXXP mutations were phosphorylated processively by Src, suggesting that individual sites are dispensable. The results also suggest that there is no defined order to the Cas phosphorylation events. We also studied the effects of these mutations by reintroducing Cas into Cas-deficient fibroblasts. Mutants lacking some or all YXXP sites augment the ability of Src to promote anchorage-independent growth. On the other hand, deletion of YXXP sites compromises the ability of Cas to promote tumor cell migration.

Variable phosphorylation states of pigment-epithelium–derived factor differentially regulate its function

AbstractThe pigment epithelium–derived factor (PEDF) belongs to the family of noninhibitory serpins. Although originally identified in the eye, PEDF is widely expressed in other body regions including the plasma. This factor can act either as a neurotrophic or as an antiangiogenic factor, and we previously showed that the 2 effects of PEDF are regulated through phosphorylation by PKA and CK2. Here, we studied the interplay between the PKA and CK2 phosphorylation of PEDF, and found that a PEDF mutant mimicking the CK2-phosphorylated PEDF cannot be phosphorylated by PKA, while the mutant mimicking the PKA-phosphorylated PEDF is a good CK2 substrate. Using triple mutants that mimic the PKA- and CK2-phosphorylated and nonphosphorylated PEDF, we found that PEDF can induce several distinct cellular activities dependent on its phosphorylation. The mutant mimicking the accumulative PKA plus CK2 phosphorylation exhibited the strongest antiangiogenic and neurotrophic activities, while the mutants mimicking the individual phosphorylation site mutants had either a reduced activity or only one of these activities. Thus, differential phosphorylation induces variable effects of PEDF, and therefore contributes to the complexity of PEDF action. It is likely that the triple phosphomimetic mutant can be used to generate effective antiangiogenic or neurotrophic drugs.

Role of Individual eNOS Phosphorylation Sites in Regulation of eNOS Activity in Endothelial Cells

Endothelial nitric oxide synthase (eNOS) catalyzes the conversion of L-arginine to L-citrulline and nitric oxide (NO). Protein phosphorylation is one of the important mechanisms for regulation of eNOS at the posttranslational level. Effects of Ser/Thr phosphorylation on eNOS conformation and activity are due to introduction of a negative charge at a specific site. Our lab’s previous work utilized Ser or Thr to Asp mutation to mimick eNOS phosphorylation, and wild-type and mutant forms of eNOS proteins were expressed and purified from a baculovirus/Sf9 cell system. To further determine the role of individual eNOS phosphorylation sites in regulation of eNOS activity in endothelial cells, in this study, we mutated all of five sites of eNOS phosphorylation to Asp or Ala. After infection of bovine aortic endothelial cells with recombinant eNOS adenoviruses, we measured eNOS activity and NO release using a cGMP reporter cell assay. We show that mimicking phosphorylation at Ser116 decreases eNOS activity compared with wild-type eNOS. Mimicking phosphorylation at Ser635 and Ser1179 at the same time does not increase eNOS activity to a greater extent than mimicking phosphorylation of either alone. In addition, removal of any of the five Ser/Thr phosphorylation sites does not affect thapsigargin- or vascular endothelial growth factor-stimulated NO release, demonstrating that maximal agonist-stimulated NO release does not require phosphorylation of any single eNOS phosphorylation site. (Supported by NIH and AHA).