Endogenous Protein Kinase Activity Research Articles

In situ study of RSK2 kinase activity in a single living cell by combining single molecule spectroscopy with activity-based probes.

Protein phosphorylation is a very important regulatory mechanism in a majority of biological processes, and the determination of protein kinase activity plays a key role in the pathological study and drug development of kinase-related diseases. However, it is very challenging to in situ study endogenous protein kinase activity in a single living cell due to the shortage of in vivo efficient methods. Here, we propose a new strategy for direct determination of protein kinase activity in a single living cell by combining single molecule fluorescence correlation spectroscopy (FCS) with activity-based probes (ABPs). Ribosomal S6 kinase-2 (RSK2) was used as a model, and the ABPs were synthesized on the basis of RSK2 inhibitor FMK to specially label active RSK2 in living cells. Conventional FCS and MEMFCS (maximum entropy method) single molecule techniques were used to in situ determine RSK2 activity in living cells based on the difference in molecular weight between free probes and probe-RSK2 complexes. Furthermore, wild-type and mutated RSK2 were fused with enhanced green fluorescent protein (EGFP) using lentivirus infection, and fluorescence cross-correlation spectroscopy (FCCS) was used to verify the selective binding of ABPs to RSK2-EGFP fusion protein in living cells. Finally, FCS with ABPs was applied for in situ monitoring of the activation of endogenous RSK2 in the stimulation of serum, epidermal growth factor, kinase inhibitors and ultraviolet irradiation; we observed that endogenous RSK2 showed different behaviors in the cytoplasm and the nucleus in some stimulation. Our results document that FCS with ABPs is a very promising method for studying endogenous protein kinases in living cells.

Long‐Wavelength Fluorescent Reporters for Monitoring Protein Kinase Activity

In vivo optical imaging must contend with the limitations imposed by the optical window of tissue (600-1000 nm). Although a wide array of fluorophores are available that are visualized in the red and near-IR region of the spectrum, with the exception of proteases, there are few long wavelength probes for enzymes. This situation poses a particular challenge for studying the intracellular biochemistry of erythrocytes, the high hemoglobin content of which optically obscures subcellular monitoring at wavelengths less than 600 nm. To address this, tunable fluorescent reporters for protein kinase activity were developed. The probing wavelength is preprogrammed by using readily available fluorophores, thereby enabling detection within the optical window of tissue, specifically in the far-red and near-IR region. These agents were used to monitor endogenous cAMP-dependent protein kinase activity in erythrocyte lysates and in intact erythrocytes when using a light-activatable reporter.

Participation of cAMP-dependent protein kinase and MAP kinase pathways during Anabas testudineus oocyte maturation

Possible involvement of cyclic nucleotide dependent protein kinase (PKA) and MAP kinase (MAPK) pathways during oocyte maturation in Anabas testudineus was investigated. Pre-incubation with phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylxanthine (IBMX), inhibited 17α, 20β-DHP-induced GVBD dose dependently. PKA inhibitor, H89 could induce resumption of meiosis independent of 17α, 20β-DHP, in dose and duration dependent manner. The maximum response was obtained with the dose of 10μM of H89 and 95% of cells underwent GVBD within 18h. Moreover, stimulation with 17α, 20β-DHP inhibited endogenous PKA activity significantly within first hour and this effect was attenuated by PDE inhibitor IBMX at all time points. The pattern of PKA inhibition corresponded well with kinetics of histone H1 kinase activation and p34cdc2 phosphorylation. These results suggest physiological relevance of cAMP/PKA signaling in perch oocytes undergoing G2/M transition. MAPK was demonstrated as two distinct isoforms (ERK1 and ERK2) which resolved in the range of 42–44kDa in immunoblot. Though total protein content did not show significant variation, H89 stimulation was able to stimulate phosphorylation of ERK1/2 from 5h onwards and the strongest response was observed between 10 and 18h. MEK inhibitor, U0126 completely blocked PKA inhibition induced MAPK activation and GVBD. In addition, inhibition of endogenous PKA by a more selective peptide inhibitor [PKI-(6–22)-amide] was sufficient to resume GVBD and MAPK activation in intact perch oocytes. Also, significant ERK1/2 phosphorylation could be stimulated in cell-free extracts of perch oocytes supplemented with PKI-(6–22)-amide. The results suggest an interaction between cAMP/PKA and MAPK pathways in mediating meiosis resumption in perch oocyte.

Up-regulation of brain-derived neurotrophic factor is regulated by extracellular signal-regulated protein kinase 5 and by nerve growth factor retrograde signaling in colonic afferent neurons in colitis

Brain-derived neurotrophic factor (BDNF) plays an essential role in sensory neuronal activation in response to visceral inflammation. Here we report that BDNF up-regulation in the primary afferent neurons in the dorsal root ganglia (DRG) in a rat model of colitis is mediated by the activation of endogenous extracellular signal-regulated protein kinase (ERK) 5 and by nerve growth factor (NGF) retrograde signaling. At 7days of colitis, the expression level of BDNF is increased in conventional neuronal tracing dye Fast Blue labeled primary afferent neurons that project to the distal colon. In these neurons, the phosphorylation (activation) level of ERK5 is also increased. In contrast, the level of phospho-ERK1/2 is not changed in the DRG during colitis. Prevention of the ERK5 activation in vivo with an intrathecal application of the MEK inhibitor PD98059 significantly attenuates the colitis-induced increases in BDNF expression in the DRG. Further studies show that BDNF up-regulation in the DRG is triggered by NGF retrograde signaling which also involves activation of the MEK/ERK pathways. Application of exogenous NGF exclusively to the compartment containing DRG nerve terminals in an ex vivo ganglia-nerve preparation markedly increases the BDNF expression level in the DRG neuronal cell body that is placed in a different compartment; this BDNF elevation is attenuated by U0126, PD98059 and a specific ERK5 inhibitor BIX02188. These results demonstrate the mechanisms and pathways by which BDNF expression is elevated in primary sensory neurons following visceral inflammation that is mediated by increased activity of ERK5 and is likely to be triggered by the elevated NGF level in the inflamed viscera.

Impaired adenosine monophosphate-activated protein kinase signalling in dorsal root ganglia neurons is linked to mitochondrial dysfunction and peripheral neuropathy in diabetes

Mitochondrial dysfunction occurs in sensory neurons and may contribute to distal axonopathy in animal models of diabetic neuropathy. The adenosine monophosphate-activated protein kinase and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) signalling axis senses the metabolic demands of cells and regulates mitochondrial function. Studies in muscle, liver and cardiac tissues have shown that the activity of adenosine monophosphate-activated protein kinase and PGC-1α is decreased under hyperglycaemia. In this study, we tested the hypothesis that deficits in adenosine monophosphate-activated protein kinase/PGC-1α signalling in sensory neurons underlie impaired axonal plasticity, suboptimal mitochondrial function and development of neuropathy in rodent models of type 1 and type 2 diabetes. Phosphorylation and expression of adenosine monophosphate-activated protein kinase/PGC-1α and mitochondrial respiratory chain complex proteins were downregulated in dorsal root ganglia of both streptozotocin-diabetic rats and db/db mice. Adenoviral-mediated manipulation of endogenous adenosine monophosphate-activated protein kinase activity using mutant proteins modulated neurotrophin-directed neurite outgrowth in cultures of sensory neurons derived from adult rats. Addition of resveratrol to cultures of sensory neurons derived from rats after 3-5 months of streptozotocin-induced diabetes, significantly elevated adenosine monophosphate-activated protein kinase levels, enhanced neurite outgrowth and normalized mitochondrial inner membrane polarization in axons. The bioenergetics profile (maximal oxygen consumption rate, coupling efficiency, respiratory control ratio and spare respiratory capacity) was aberrant in cultured sensory neurons from streptozotocin-diabetic rats and was corrected by resveratrol treatment. Finally, resveratrol treatment for the last 2 months of a 5-month period of diabetes reversed thermal hypoalgesia and attenuated foot skin intraepidermal nerve fibre loss and reduced myelinated fibre mean axonal calibre in streptozotocin-diabetic rats. These data suggest that the development of distal axonopathy in diabetic neuropathy is linked to nutrient excess and mitochondrial dysfunction via defective signalling of the adenosine monophosphate-activated protein kinase/PGC-1α pathway.

TTBK2 kinase substrate specificity and the impact of spinocerebellar-ataxia-causing mutations on expression, activity, localization and development.

Mutations that truncate the C-terminal non-catalytic moiety of TTBK2 (tau tubulin kinase 2) cause the inherited, autosomal dominant, SCA11 (spinocerebellar ataxia type11) movement disorder. In the present study we first assess the substrate specificity of TTBK2 and demonstrate that it has an unusual preference for a phosphotyrosine residue at the +2 position relative to the phosphorylation site. We elaborate a peptide substrate (TTBKtide, RRKDLHDDEEDEAMSIYpA) that can be employed to quantify TTBK2 kinase activity. Through modelling and mutagenesis we identify a putative phosphate-priming groove within the TTBK2 kinase domain. We demonstrate that SCA11 truncating mutations promote TTBK2 protein expression, suppress kinase activity and lead to enhanced nuclear localization. We generate an SCA11-mutation-carrying knockin mouse and show that this leads to inhibition of endogenous TTBK2 protein kinase activity. Finally, we find that, in homozygosity, the SCA11 mutation causes embryonic lethality at embryonic day 10. These findings provide the first insights into some of the intrinsic properties of TTBK2 and reveal how SCA11-causing mutations affect protein expression, catalytic activity, localization and development. We hope that these findings will be helpful for future investigation of the regulation and function of TTBK2 and its role in SCA11.

Differential Regulation of Proteasome Function in Isoproterenol-Induced Cardiac Hypertrophy

Proteasomal degradation is altered in many disease phenotypes including cardiac hypertrophy, a prevalent condition leading to heart failure. Our recent investigations identified heterogeneous subpopulations of proteasome complexes in the heart and implicated multiple mechanisms for their regulation. The study aimed at identification of molecular mechanisms changing proteasome function in the hypertrophic heart. Proteasome function, expression, and assembly were analyzed during the development of cardiac hypertrophy induced by β-adrenergic stimulation. The analysis revealed, for the first time, divergent regulation of proteasome function in cardiac hypertrophy. Proteasome complexes have 3 different proteolytic activities, which are ATP-dependent for 26S complexes (19S assembled with 20S) and ATP-independent for 20S core particles. The 26S activities were enhanced in hypertrophic hearts, partially because of increased expression and assembly of 19S subunits with 20S core complexes. In contrast, caspase- and trypsin-like 20S activities were significantly decreased. Activation of endogenous cAMP-dependent protein kinase (PKA) rescued the depressed 20S functions, supporting the notion that PKA signaling is a positive regulator of protein degradation in the heart. Chymotrypsin-like 20S activity was stably maintained during cardiac remodeling, indicating a switch in proteasome subpopulations, which was supported by altered expression and incorporation of inducible β subunits. Three novel mechanisms for the regulation of proteasome activities were discovered in the development of cardiac hypertrophy: (1) increased incorporation of inducible subunits in 20S proteasomes; (2) enhanced 20S sensitivity to PKA activation; and (3) increased 26S assembly. PKA modulation of proteasome complexes may provide a novel therapeutic avenue for restoration of cardiac function in the diseased myocardium.

Substrate specificity and inhibitors of LRRK2, a protein kinase mutated in Parkinson's disease

The LRRK2 (leucine-rich repeat protein kinase-2) is mutated in a significant number of Parkinson's disease patients, but little is known about its regulation and function. A common mutation changing Gly2019 to serine enhances catalytic activity, suggesting that small-molecule inhibitors might have utility in treating Parkinson's disease. We employed various approaches to explore the substrate-specificity requirements of LRRK2 and elaborated a peptide substrate termed Nictide, that had 20-fold lower Km and nearly 2-fold higher Vmax than the widely deployed LRRKtide substrate. We demonstrate that LRRK2 has marked preference for phosphorylating threonine over serine. We also observed that several ROCK (Rho kinase) inhibitors such as Y-27632 and H-1152, suppressed LRRK2 with similar potency to which they inhibited ROCK2. In contrast, GSK429286A, a selective ROCK inhibitor, did not significantly inhibit LRRK2. We also identified a mutant LRRK2[A2016T] that was normally active, but resistant to H-1152 and Y-27632, as well as sunitinib, a structurally unrelated multikinase inhibitor that, in contrast with other compounds, suppresses LRRK2, but not ROCK. We have also developed the first sensitive antibody that enables measurement of endogenous LRRK2 protein levels and kinase activity as well as shRNA (short hairpin RNA) methods to reduce LRRK2 expression. Finally, we describe a pharmacological approach to validate whether substrates are phosphorylated by LRRK2 and use this to provide evidence that LRRK2 may not be rate-limiting for the phosphorylation of the proposed substrate moesin. The findings of the present study will aid with the investigation of LRRK2.

Maize Plasma Membrane Aquaporins Belonging to the PIP1 and PIP2 Subgroups are in vivo Phosphorylated

Aquaporins are channel proteins that facilitate transmembrane water movement. In this study, we showed that plasma membrane intrinsic proteins (PIPs) from maize shoots are in vitro and in vivo phosphorylated on serine residues by a calcium-dependent kinase associated with the membrane fraction. Mass spectrometry identified phosphorylated peptides corresponding to the C-terminal region of (i) ZmPIP2;1, ZmPIP2;2 and/or ZmPIP2;7; (ii) ZmPIP2;3 and/or ZmPIP2;4; (iii) ZmPIP2;6; together with (iv) a phosphorylated peptide located in the N-terminal region of ZmPIP1;1, ZmPIP1;2, ZmPIP1;3 and/or ZmPIP1;4. The role of phosphorylation in the water channel activity of wild-type and mutant ZmPIP2;1 was studied in Xenopus laevis oocytes. Activation of endogenous protein kinase A increased the osmotic water permeability coefficient of ZmPIP2;1-expressing oocytes, suggesting that phosphorylation activates its channel activity. Mutation of S126 or S203, putative phosphorylated serine residues conserved in all plant PIPs, to alanine decreased ZmPIP2;1 activity by 30-50%, without affecting its targeting to the plasma membrane. Mutation of S285, which is phosphorylated in planta, to alanine or glutamate did not affect the water channel activity. These results indicate that, in oocytes, S126 and S203 play an important role in ZmPIP2;1 activity and that phosphorylation of S285 is not required for its activity.

Protein Kinase A, not Epac, Suppresses Hedgehog Activity and Regulates Glucocorticoid Sensitivity in Acute Lymphoblastic Leukemia Cells

Cyclic AMP synergizes strongly with glucocorticoids (GC) to induce apoptosis in normal or malignant lymphoid cells. We examined the individual roles that cAMP-dependent protein kinase (PKA) and Epac (exchange protein directly activated by cAMP), two intracellular cAMP receptors, play in this synergistic effect. Our studies demonstrate that PKA is responsible for the observed synergism with GC, whereas Epac exerts a weak antagonistic effect against GC-induced apoptosis. We find that endogenous PKA activity is higher in the GC-sensitive clone than in the GC-resistant clone. In the GC-sensitive clone, higher PKA activity is associated with lower Hedgehog (Hh) activity. Moreover, inhibition of Hh activity by Hh pathway-specific inhibitors leads to cell cycle arrest and apoptosis in CEM (human acute lymphoblastic leukemia, T lineage) cells, and the GC-sensitive clone is more sensitive to Hh inhibition. These results suggest that Hh activity is critical for leukemia cell growth and survival and that the level of Hh activity is in part responsible for the synergism between cAMP and GC.

Protein kinase inhibitors reduce GABA but not glutamate release in the nucleus accumbens

We investigated the role of endogenous protein kinase activity on synaptic transmission in the rat nucleus accumbens slice. The isoquinolinesulfonamide H-7 (50 μM), a non-selective serine/threonine protein kinase inhibitor, had no effect on pharmacologically isolated glutamatergic EPSCs. However, it reduced GABA release in a dose-dependent manner. This effect of H-7 was not mimicked by the selective cAMP-dependent protein kinase inhibitor H-89, the PKC inhibitor Bisindolylmaleimide-1, or the cGMP-dependent protein kinase inhibitor KT5823. However, bath application of the myosin light chain kinase (MLCK) inhibitor, ML-7, significantly reduced IPSC amplitudes and partially occluded the reduction in IPSCs observed following bath application of H-7. These results suggest that endogenous protein kinase activity, specifically MLCK activity, regulates GABA, but not glutamate release, onto medium spiny neurons in the nucleus accumbens.

Modulation of Ca2+-activated K+ channels of human erythrocytes by endogenous protein kinase C

Single IKCa channels of human erythrocytes were studied with the patch-clamp technique to define their modulation by endogenous protein kinase C (PKC). The perfusion of the cytoplasmic side of freshly excised patches with the PKC activator, phorbol 12-myristate 13-acetate (PMA), inhibited channel activity. This effect was blocked by PKC19–31, a peptide inhibitor specific for PKC. Similar results were obtained by perfusing the membrane patches with the structurally unrelated PKC activator 1-oleoyl-2-acetylglycerol (OAG). Blocking of this effect was induced by perfusion with PKC19–31 or chelerythrine. Channel activity was not inhibited by the PMA analog 4α-phorbol 12,13-didecanoate (4αPDD), which has no effect on PKC. Activation of endogenous cAMP-dependent protein kinase (PKA), which is known to up-modulate IKCa channels, restored channel activity previously inhibited by OAG. The application of OAG induced a reversible reduction of channel activity previously up-modulated by the activation of PKA, indicating that the effects of the two kinases are commutative, and antagonistic. Kinetic analysis showed that down-regulation by PKC mainly changes the opening frequency without significantly affecting mean channel open time and conductance. These results provide evidence that an endogenous PKC down-modulates the activity of native IKCa channels of human erythrocytes. Our results show that PKA and PKC signal transduction pathways integrate their effects, determining the open probability of the IKCa channels.

Rac1-MKK3-p38-MAPKAPK2 Pathway Promotes Urokinase Plasminogen Activator mRNA Stability in Invasive Breast Cancer Cells

We reported previously that down-regulating or functionally blocking alphav integrins inhibits endogenous p38 mitogen-activated protein kinase (MAPK) activity and urokinase plasminogen activator (uPA) expression in invasive MDA-MB-231 breast cancer cells whereas engaging alphav integrins with vitronectin activates p38 MAPK and up-regulates uPA expression (Chen, J., Baskerville, C., Han, Q., Pan, Z., and Huang, S. (2001) J. Biol. Chem. 276, 47901-47905). Currently, it is not clear what upstream and downstream signaling molecules of p38 MAPK mediate alphav integrin-mediated uPA up-regulation. In the present study, we found that alphav integrin ligation activated small GTPase Rac1 preferentially, and dominant negative Rac1 inhibited alphav integrin-mediated p38 MAPK activation. Using constitutively active MAPK kinases, we found that both constitutively active MKK3 and MKK6 mutants were able to activate p38 MAPK and up-regulate uPA expression, but only dominant negative MKK3 blocked alphav integrin-mediated p38 MAPK activation and uPA up-regulation. These results suggest that MKK3, rather than MKK6, mediates alphav integrin-induced p38 MAPK activation. Among the potential downstream effectors of p38 MAPK, we found that only MAPK-activated protein kinase 2 affects alphav integrin-mediated uPA up-regulation significantly. Finally, using beta-globin reporter gene constructs containing uPA mRNA 3'-untranslated region (UTR) and adenosine/uridine-rich elements-deleted 3'-UTR, we demonstrated that p38 MAPK/MAPK-activated protein kinase 2 signaling pathway regulated uPA mRNA stability through a mechanism involving the adenosine/uridine-rich elements sequence in 3'-UTR of uPA mRNA.

Depressed levels of Ca2+-cycling proteins may underlie sarcoplasmic reticulum dysfunction in the diabetic heart.

In view of the depressed sarcoplasmic reticulum (SR) Ca2+-pump and Ca2+-release activities in the diabetic heart and the critical role of phosphorylation in regulating the SR function, we examined the status of Ca2+-calmodulin-dependent protein kinase (CaMK) and cAMP-dependent protein kinase (PKA)-mediated phosphorylations in the diabetic heart. Diabetes was induced in male Sprague-Dawley rats by an injection of streptozotocin (65 mg/kg i.v.), and the animals were killed 6 weeks later for assessment of the ventricular SR function. Depressed cardiac performance and SR Ca2+-uptake and -release activities in diabetic animals were accompanied by a significant decrease in the level of SR Ca2+-cycling proteins, such as ryanodine receptor, Ca2+-pump ATPase, and phospholamban. On the other hand, the CaMK- and PKA-mediated phosphorylations of these Ca2+-cycling proteins, the endogenous SR CaMK and PKA activities, and the endogenous SR and cytosolic phosphatase activities were increased in the diabetic heart. Treatment of 3-week diabetic animals with insulin partially or fully prevented the diabetes-induced changes in cardiac performance, SR Ca2+-uptake and -release activites, and SR protein content, whereas the diabetes-induced changes in SR CaMK- and PKA-mediated phosphorylations and activities, as well as phosphatase activities, were not significantly affected. These results suggest that the reduced content of the Ca2+-cycling proteins, unlike alterations in PKA and phosphatase activities, appear to be the major defect underlying SR dysfunction in the diabetic heart.

A Catalytically Inactive Mutant of Type I cGMP-dependent Protein Kinase Prevents Enhancement of Large Conductance, Calcium-sensitive K+ Channels by Sodium Nitroprusside and cGMP

The activation of large conductance, calcium-sensitive K(+) (BK(Ca)) channels by the nitric oxide (NO)/cyclic GMP (cGMP) signaling pathway appears to be an important cellular mechanism contributing to the relaxation of smooth muscle. In HEK 293 cells transiently transfected with BK(Ca) channels, we observed that the NO donor sodium nitroprusside and the membrane-permeable analog of cGMP, dibutyryl cGMP, were both able to enhance BK(Ca) channel activity 4-5-fold in cell-attached membrane patches. This enhancement correlated with an endogenous cGMP-dependent protein kinase activity and the presence of the alpha isoform of type I cGMP-dependent protein kinase (cGKI). We observed that co-transfection of cells with BK(Ca) channels and a catalytically inactive ("dead") mutant of human cGKIalpha prevented enhancement of BK(Ca) channel in response to either sodium nitroprusside or dibutyryl cGMP in a dominant negative fashion. In contrast, expression of wild-type cGKIalpha supported enhancement of channel activity by these two agents. Importantly, both endogenous and expressed forms of cGKIalpha were found to associate with BK(Ca) channel protein, as demonstrated by a reciprocal co-immunoprecipitation strategy. In vitro, cGKIalpha was able to directly phosphorylate immunoprecipitated BK(Ca) channels, suggesting that cGKIalpha-dependent phosphorylation of BK(Ca) channels in situ may be responsible for the observed enhancement of channel activity. In summary, our data demonstrate that cGKIalpha alone is sufficient to promote the enhancement of BK(Ca) channels in situ after activation of the NO/cGMP signaling pathway.

Protein Kinase A Associates with Cystic Fibrosis Transmembrane Conductance Regulator via an Interaction with Ezrin

The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial Cl(-) channel whose activity is controlled by cAMP-dependent protein kinase (PKA)-mediated phosphorylation. We found that CFTR immunoprecipitates from Calu-3 airway cells contain endogenous PKA, which is capable of phosphorylating CFTR. This phosphorylation is stimulated by cAMP and inhibited by the PKA inhibitory peptide. The endogenous PKA that co-precipitates with CFTR could also phosphorylate the PKA substrate peptide, Leu-Arg-Arg-Ala-Ser-Leu-Gly (kemptide). Both the catalytic and type II regulatory subunits of PKA are identified by immunoblotting CFTR immunoprecipitates, demonstrating that the endogenous kinase associated with CFTR is PKA, type II (PKA II). Phosphorylation reactions mediated by CFTR-associated PKA II are inhibited by Ht31 peptide but not by the control peptide Ht31P, indicating that a protein kinase A anchoring protein (AKAP) is responsible for the association between PKA and CFTR. Ezrin may function as this AKAP, since it is expressed in Calu-3 and T84 epithelia, ezrin binds RII in overlay assays, and RII is immunoprecipitated with ezrin from Calu-3 cells. Whole-cell patch clamp of Calu-3 cells shows that Ht31 peptide reduces cAMP-stimulated CFTR Cl(-) current, but Ht31P does not. Taken together, these data demonstrate that PKA II is linked physically and functionally to CFTR by an AKAP interaction, and they suggest that ezrin serves as an AKAP for PKA-mediated phosphorylation of CFTR.

Polarized sphingolipid transport from the subapical compartment changes during cell polarity development.

The subapical compartment (SAC) plays an important role in the polarized transport of proteins and lipids. In hepatoma-derived HepG2 cells, fluorescent analogues of sphingomyelin and glucosylceramide are sorted in the SAC. Here, evidence is provided that shows that polarity development is regulated by a transient activation of endogenous protein kinase A and involves a transient activation of a specific membrane transport pathway, marked by the trafficking of the labeled sphingomyelin, from the SAC to the apical membrane. This protein kinase A-regulated pathway differs from the apical recycling pathway, which also traverses SAC. After reaching optimal polarity, the direction of the apically activated pathway switches to one in the basolateral direction, without affecting the apical recycling pathway.

In vitro phosphorylation of high molecular weight glutenin subunits from wheat endosperm

We have investigated the in vitro phosphorylation of high molecular weight glutenin subunits (HMW-GS), a group of non-soluble proteins present in wheat endosperm. Computer aided searches of potential biological sites in the known sequences of these proteins have evidenced the presence of sequence motifs specific for protein kinase C (PKC), calcium-dependent protein kinase from wheat, casein kinase II, tyrosine protein kinase and glycosylation. We have demonstrated that subunit 1Bx7 is a substrate of a partially purified PKC from rat brain. Further experiments have shown that this subunit is phosphorylated by an endogenous protein kinase activity found in wheat flour. These preliminary results are important for the possible implications on the structure-function relationships of these proteins and could probably suggest, for the first time, a potential physiological role in particular situations for some HMW-GS.

Endogenous casein kinase I catalyzes the phosphorylation of the lens fiber cell connexin49.

The lens fiber cell-specific gap junction protein connexin49 is a substrate for a membrane-associated Ser/Thr protein kinase that can be extracted from lens cell membranes by 0.6 M KCl. However, the identity of this protein kinase has not been defined. In this report, evidence is presented indicating that it is casein kinase I. Thus, connexin49 was shown to be a substrate for purified casein kinase I but not for casein kinase II; the endogenous connexin49 protein kinase activity extracted from lens membranes with KCl was inhibited by the casein kinase I-specific inhibitor, N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide (CKI-7); the connexin49 protein kinase activity in the lens membrane KCl extract, which could be partially purified by gel filtration and affinity purification with a casein-Sepharose 4B column, copurified with casein kinase activity; phosphopeptide analysis showed that casein kinase I and the connexin49 protein kinase activity in the lens membrane KCl extract probably share the same phosphorylation sites in connexin49. Reverse transcription-PCR using total ovine lens RNA and casein kinase I isoform-specific oligonucleotide primers resulted in the amplification of cDNAs encoding casein kinase I-alpha and -gamma, while an in-gel casein kinase assay indicated casein kinase activity in the lens membrane KCl extract was associated with a major 39.2-kDa species, which is consistent with the 36 to 40-kDa size of casein kinase I-alpha in other animal species. These results demonstrate that the protein kinase activity present in the lens membrane 0.6 M KCl extract that catalyzes the phosphorylation of connexin49 is casein kinase I, probably the alpha isoform.

BRCA1 is phosphorylated at serine 1497 in vivo at a cyclin-dependent kinase 2 phosphorylation site.

BRCA1 is a cell cycle-regulated nuclear protein that is phosphorylated mainly on serine and to a lesser extent on threonine residues. Changes in phosphorylation occur in response to cell cycle progression and DNA damage. Specifically, BRCA1 undergoes hyperphosphorylation during late G1 and S phases of the cell cycle. Here we report that BRCA1 is phosphorylated in vivo at serine 1497 (S1497), which is part of a cyclin-dependent kinase (CDK) consensus site. S1497 can be phosphorylated in vitro by CDK2-cyclin A or E. BRCA1 coimmunoprecipitates with an endogenous serine-threonine protein kinase activity that phosphorylates S1497 in vitro. This cellular kinase activity is sensitive to transfection of a dominant negative form of CDK2 as well as the application of the CDK inhibitors p21 and butyrolactone I but not p16. Furthermore, BRCA1 coimmunoprecipitates with CDK2 and cyclin A. These results suggest that the endogenous kinase activity is composed of CDK2-cyclin complexes, at least in part, concordant with the G1/S-specific increase in BRCA1 phosphorylation.