Proline-directed Protein Kinase Research Articles

Identification of Phosphorylation Sites in the Translational Regulator, PHAS-I, That Are Controlled by Insulin and Rapamycin in Rat Adipocytes

Phosphorylation of PHAS-I by mitogen-activated protein (MAP) kinase in vitro decreased PHAS-I binding to eukaryotic initiation factor (eIF)-4E. The decrease in binding lagged behind the phosphorylation of PHAS-I in Ser64, the preferred site of MAP kinase. Binding of the Ala64 mutant of PHAS-I to eIF-4E was abolished by MAP kinase, indicating that phosphorylation of sites other than Ser64 control binding. To identify such sites, PHAS-I was phosphorylated with MAP kinase and [gamma-32P]ATP and then cleaved proteolytically before the resulting phosphopeptides were isolated by reverse phase chromatography and directly identified by amino acid sequencing. Phosphorylated residues were located by determining the cycles in which 32P was released when phosphopeptides were subjected to sequential Edman degradation. With an extended incubation in vitro, MAP kinase phosphorylated Thr36, Thr45, Ser64, Thr69, and Ser82. In rat adipocytes, the phosphorylation of all five sites was increased by insulin and decreased by rapamycin although there were differences in the magnitude of the effects. A form of PHAS-I phosphorylated exclusively in Thr36 remained bound to eIF-4E, indicating that phosphorylation of Thr36 is insufficient for dissociation of the PHAS-I.eIF-4E complex. In summary, our results indicate that multiple phosphorylation sites are involved in the control of PHAS-I. All five sites identified fit a (Ser/Thr)-Pro motif, suggesting that the phosphorylation of PHAS-I in cells is mediated by a proline-directed protein kinase.

Reversible tyrosine phosphorylation/dephosphorylation of proline-directed protein kinase FA/glycogen synthase kinase-3α in A431 cells

Modulation of protein kinase FA/glycogen synthase kinase-3alpha (kinase FA/GSK-3alpha) by reversible tyrosine phosphorylation/dephosphorylation was investigated. In addition to genistein, other protein tyrosine kinase (PTK) inhibitors, such as tyrphostin A47 and B42, also could induce tyrosine dephosphorylation and inactivation of kinase FA/GSK-3alpha in A431 cells, and this process was found to be reversible. Pretreatment of the cells with 100 microM orthovanadate, a protein tyrosine phosphatase (PTP) inhibitor, could diminish significantly the effects of PTK inhibitors on both enzyme activity and phosphotyrosine content of the kinase, suggesting that the PTK inhibitors induced tyrosine dephosphorylation/inactivation of this kinase is mediated by orthovanadate-sensitive PTP(s) in A431 cells. Moreover, the phosphotyrosine moiety of kinase FA/GSK-3alpha was found to be highly turned over in resting cells. Interestingly, we found that the less active, tyrosine-dephosphorylated form of kinase FA/GSK-3alpha immunoprecipitated from genistein-treated cells was able to reactivate partially with concomitant rephosphorylation of tyrosine residue in vitro. Taken together, these findings demonstrate that tyrosine phosphorylation and concomitant activation of kinase FA/GSK-3alpha can be carried out both in vitro and in vivo and an in vivo phosphatase activity may function in antagonism to PTK activation of kinase FA/GSK-3alpha.

P-448 - Phosphorylation of microtubule-associated protein 2 by proline-directed protein kinases in cerebellar culture cells

P-448 - Phosphorylation of microtubule-associated protein 2 by proline-directed protein kinases in cerebellar culture cells

Okadaic acid induces hyperphosphorylation of tau independently of mitogen-activated protein kinase activation.

Hyperphosphorylation of the microtubule-associated protein tau is a characteristic of Alzheimer brain tissue. Recent in vitro data suggest that mitogen-activated protein kinase (MAPK), a proline-directed protein kinase, phosphorylates the sites on tau common to Alzheimer's disease. Using an okadaic acid-induced tau hyperphosphorylation model, we have tested the requirement for MAPK activity, using a specific inhibitor ¿PD098059 [2-(2'-amino-3'-methoxyphenyl)oxanaphthalen-4-one]¿ of the MAPK activator Mek1. Mobility shift, phosphoepitope analysis, and direct measurement of kinase activity indicated that the Mek1 inhibitor dose-dependently blocked basal and okadaic acid-induced MAPK activation. Despite a block of MAPK activation by this inhibitor, robust tau hyperphosphorylation was observed in response to okadaic acid. In addition, activation of MAPK by phorbol 12-myristate 13-acetate did not result in tau phosphorylation, indicating that in primary cultures of cortical neurons elevated MAPK activity is not sufficient to induce tau hyperphosphorylation.

Distribution, levels, and activity of glycogen synthase kinase-3 in the Alzheimer disease brain.

A number of studies have implicated a proline-directed protein kinase, glycogen synthase kinase-3 (GSK-3) in the hyperphosphorylation of tau in Alzheimer's disease (AD). Toward understanding the role of GSK-3 in the abnormal hyperphosphorylation of tau in AD we have found that GSK-3 is prominently present in neuronal cell bodies and their processes and co-localizes with neurofibrillary changes in AD brain. Furthermore, the levels of GSK-3 as determined by indirect ELISA are approximately 50% increased in the postsynaptosomal supernatant from AD brains as compared to the controls. However, no increase in GSK-3 enzyme activity was detected. In AD brain, with its reduced phosphatase activity, even normal levels of GSK-3 activity might be sufficient for the hyperphosphorylation of tau.

Phosphorylation and dephosphorylation in the proline-rich C-terminal domain of microtubule-associated protein 2.

The C-terminal domain of microtubule-associated protein 2 (MAP2) contains a proline-rich region and the tubulin-binding domain. We have generated antibodies to follow the phosphorylation state of the proline-rich domain. One of these antibodies (no. 305) has been raised against a synthetic peptide P (sequence RTPGTPGTPSY) phosphorylated at the threonine residues. This sequence is present in the proline-rich region of MAP2 and is phosphorylated in vitro by at least three different proline-directed protein kinases: p42mpk, p34cdc2, and GSK3 (glycogen-synthase kinase 3) alpha/beta. The MAP2 sites phosphorylated by these kinases are different, although all of them phosphorylate the C-terminal domain of MAP2 as determined by Staphylococcus aureus V8 protease mapping. Nonphosphorylated peptide P can be phosphorylated in vitro by all three kinases studied with similar efficiency. In high-molecular-mass MAP2, this sequence is highly phosphorylated in vivo at the late stages of rat development. This motif can be rapidly dephosphorylated in vitro by protein-phosphatase 1 (PP1) and 2A (PP2A) catalytic subunits but not by PP2B.

In vitro phosphorylation of the cytoplasmic domain of the amyloid precursor protein by glycogen synthase kinase-3beta.

The two pathological lesions found in the brains of Alzheimer's disease patients, neurofibrillary tangles and neuritic plaques, are likely to be formed through a common pathway. Neurofibrillary tangles are intracellular aggregates of paired helical filaments, the main component of which is hyperphosphorylated forms of the microtubule-associated protein tau. Extracellular neuritic plaques and diffuse and vascular amyloid deposits are aggregates of beta-amyloid protein, a 4-kDa protein derived from the amyloid precursor protein (APP). Using conditions in vitro under which two proline-directed protein kinases, glycogen synthase kinase-3beta (GSK-3beta) and mitogen-activated protein kinase (MAPK), were able to hyperphosphorylate tau, GSK-3beta but not MAPK phosphorylated recombinant APPcyt. The sole site of phosphorylation in APPcyt by GSK-3beta was determined by phosphoamino acid analysis and phosphorylation of APPcyt mutant peptides to be Thr743 (numbering as for APP770). This site was confirmed by endoproteinase Glu-C digestion of APPcyt and peptide sequencing. The ability of GSK-3beta to phosphorylate APPcyt and tau provides a putative link between the two lesions and indicates a critical role of GSK-3beta in the pathogenesis of Alzheimer's disease.

Site-specific Phosphorylation of Synapsin I by Mitogen-activated Protein Kinase and Cdk5 and Its Effects on Physiological Functions

Posttranslational modifications of synapsin I, a major phosphoprotein in synaptic terminals, were studied by mass spectrometry. In addition to a well known phosphorylation site by calmodulin-dependent protein kinase II (CaM kinase II), a hitherto unrecognized site (Ser553) was found phosphorylated in vivo. The phosphorylation site is immediately followed by a proline, suggesting that the protein is an in vivo substrate of so-called proline-directed protein kinase(s). To identify the kinase involved, three proline-directed protein kinases expressed highly in the brain, i.e. mitogen-activated protein (MAP) kinase, Cdk5-p23, and glycogen synthase kinase 3beta, were tested for the in vitro phosphorylation of synapsin I. Only MAP kinase and Cdk5-p23 phosphorylated synapsin I stoichiometrically. The phosphorylation sites were determined to be Ser551 and Ser553 with Cdk5-p23, and Ser62, Ser67, and Ser551 with MAP kinase. Upon phosphorylation with MAP kinase, synapsin I showed reduced F-actin bundling activity, while no significant effect on the interaction was observed with the protein phosphorylated with Cdk5-p23. These results raise the possibility that the so-called proline-directed protein kinases together with CaM kinase II and cAMP-dependent protein kinase play an important role in the regulation of synapsin I function.

Mimosine Blocks Cell Cycle Progression by Chelating Iron in Asynchronous Human Breast Cancer Cells

Mimosine is a toxic nonprotein amino acid that is a major constituent of the tropical legumesLeucaenaandMimosa.Mimosine has been shown to cause acute and chronic toxicosis in livestock fed from forage containing these plants. Recently, mimosine has been demonstrated to reversibly block cell cycle progression in mammalian cells in culture. In this study, we compared the effects of mimosine to desferrioxamine (DFO), a well-characterized iron chelator, and found that both chemicals similarly altered cell cycle progression in MDA-MB-453 human breast cancer cells. Mimosine (400 μm) and DFO (150 μm) both reduced DNA synthesis by greater than 90% of control within 4 hr of treatment, and suppressed total proline-directed protein kinase activity to less than 10% of control after 16 hr treatment. These effects were antagonized by the addition of iron as ferrous sulfate (250 μm), which is bound to transferrin and imported into the cell via transferrin receptor endocytosis, or as hemin (100 μm), which passes through the cell membrane and releases iron into the cytosol. After 24 hr treatment with the chelators, a large portion of the available transferrin receptors moved to the cell surface, indicating that the cells were iron-starved. Our data demonstrate that mimosine, through iron chelation, blocks cell cycle progression in MDA-MB-453 human breast cancer cells.

Dephosphorylated but not phosphorylated microtubule associated protein MAP1B binds to microfilaments

We have reported that purified native MAP1B interacts with microtubules but not with microfilaments [Pedrotti and Islam, Cell Motil. Cytoskel. (1995) 30, 301–309]. However, MAP1B can be phosphorylated at multiple sites by casein kinase II (CKII) and proline-directed protein kinases (PDPK) and immunoblotting studies show that purified native MAP1B is phosphorylated at least at two CKII sites and at one PDPK site [Pedrotti et al., Biochemistry (1996) 35, 3016–3023]. We now show that phosphorylation affects the in vitro binding of MAP1B with microfilaments. Native MAP1B does not bind to microfilaments but after treatment with alkaline phosphatase the dephosphorylated MAP1B binds and cosediments with microfilaments. Dephosphorylation kinetics suggest that the PDPK site, but not CKII sites, may negatively regulate the interaction with F-actin. The ability of dephosphorylated MAP1B to crosslink microfilaments was also examined and showed that MAP1B exhibits only a weak crosslinking of F-actin when compared with MAP2.

A Novel Cytoplasmic Domain of the p55 Tumor Necrosis Factor Receptor Initiates the Neutral Sphingomyelinase Pathway

The human p55 tumor necrosis factor (TNF) receptor (TR55) initiates at least two independent signaling cascades. The acidic sphingomyelinase (A-SMase) pathway involves a phosphatidylcholine-specific phospholipase C, an endosomal A-SMase, and controls expression of multiple TNF-responsive genes through induction of transcription factors such as NF-kappaB. The neutral sphingomyelinase (N-SMase) pathway comprises a membrane-bound N-SMase, proline-directed protein kinases, as well as phospholipase A2 and appears critical for the inflammatory responses induced by TNF. While the domain of TR55 that induces A-SMase is probably identical to the death domain, the exact location and extent of a putative N-SMase activation domain are still unknown. Structure-function analysis of TR55 deletion mutants revealed a novel region of 11 amino acids at position 309-319 that is both necessary and sufficient for activation of N-SMase. The N-SMase activation domain is distinct from the death domain and incapable of induction of A-SMase, NF-kappaB, and cytotoxicity. Taken together, our results suggest that a functionally independent region of TR55 is responsible for selectively initiating the N-SMase pathway that couples to an important inflammatory signaling cascade.

Overexpression of protein kinase FA/GSK-3α (a proline-directed protein kinase) correlates with human hepatoma dedifferentiation/progression

Overexpression of protein kinase FA/GSK-3α (a proline-directed protein kinase) correlates with human hepatoma dedifferentiation/progression

Evidence for Phosphorylation of CTP:Phosphocholine Cytidylyltransferase by Multiple Proline-directed Protein Kinases

Reversible phosphorylation of CTP:phosphocholine cytidylyltransferase, the rate-limiting enzyme of phosphatidylcholine biosynthesis, is thought to play a role in regulating its activity. In the present study, the hypothesis that proline-directed kinases play a major role in phosphorylating cytidylyltransferase is substantiated using a c-Ha-ras-transfected clone of the human keratinocyte cell line HaCaT. Cellular extracts from epidermal growth factor-stimulated HaCaT cells and from ras-transfected HaCaT cells phosphorylated cytidylyltransferase much stronger as compared with extracts from quiescent HaCaT cells. The tryptic phosphopeptide pattern of cytidylyltransferase phosphorylated by cell-free extracts from ras-transfected HaCaT cells was similar compared with the patterns of cytidylyltransferase phosphorylated by p44mpkmitogen-activated protein kinase and p34cdc2 kinase in vitro, whereas in the case of casein kinase II the pattern was different. Furthermore, in c-Ha-ras-transfected HaCaT cells the in vivo phosphorylation state of cytidylyltransferase was 2-fold higher as compared with untransfected HaCaT cells. This higher phosphorylation of cytidylyltransferase in the ras-transfected clone was reduced to a level below the phosphorylation of cytidylyltransferase in untransfected cells, using olomoucine, a specific inhibitor of proline-directed kinases. The reduced phosphorylation of cytidylyltransferase in olomoucine-treated cells correlated with an enhanced stimulation of enzyme activity by oleic acid.

Sendai Virus P Protein Is Constitutively Phosphorylated at Serine249: High Phosphorylation Potential of the P Protein

Previously we showed that the Sendai virus P protein (568 aa) in virus-infected cells and in virions was primarily and constitutively phosphorylated on serine(s) in a single tryptic phosphopeptide TP1. By two-dimensional thin-layer electrophoresis and chromatography analysis of tryptic phosphopeptides of several deletion and point mutants of the P protein, we now show that the sole phosphorylation site in TP1 is serine249. Interestingly, when serine249 was deleted or mutagenized alternate potential serine sites were more heavily phosphorylated. A similar effect was observed when the deletion was very close to serine249 (Δ208–236). Mutagenesis of proline250 to alanine abrogated phosphorylation at serine249 suggesting that proline250 is essential for the primary phosphorylation of the P protein. Conceivably, serine249 phosphorylation is mediated by a proline-directed protein kinase. This finding is unusual because a majority of the P proteins from other negative-strand RNA viruses have been shown to be phosphorylated primarily by casein kinase II. Our results demonstrate that the P protein has a strong potency to remain phosphorylated. Based on our previous and present results, we suggest that the phosphorylation sites on P are dependent on the accessibility of phosphatases rather than kinases as all potential sites are about equally competent for phosphorylation. We propose that phosphorylation is important for maintaining the structural integrity of the Sendai virus P protein.

GTPase-Deficient Gα16 and Gαq Induce PC12 Cell Differentiation and Persistent Activation of cJun NH2-Terminal Kinases

Persistent stimulation of specific protein kinase pathways has been proposed as a key feature of receptor tyrosine kinases and intracellular oncoproteins that signal neuronal differentiation of rat pheochromocytoma (PC12) cells. Among the protein serine/threonine kinases identified to date, the p42/44 mitogen-activated protein (MAP) kinases have been highlighted for their potential role in signalling PC12 cell differentiation. We report here that retrovirus-mediated expression of GTPase-deficient, constitutively active forms of the heterotrimeric Gq family members, G alpha qQ209L and G alpha 16Q212L, in PC12 cells induces neuronal differentiation as indicated by neurite outgrowth and the increased expression of voltage-dependent sodium channels. Differentiation was not observed after cellular expression of GTPase-deficient forms of alpha i2 or alpha 0, indicating selectivity for the Gq family of G proteins. As predicted, overexpression of alpha qQ209L and alpha 16Q212L constitutively elevated basal phospholipase C activity approximately 10-fold in PC12 cells. Significantly, little or no p42/44 MAP kinase activity was detected in PC12 cells differentiated with alpha 16Q212L or alpha qQ209L, although these proteins were strongly activated following expression of constitutively active cRaf-1. Rather, a persistent threefold activation of the cJun NH2-terminal kinases (JNKs) was observed in PC12 cells expressing alpha qQ209L and alpha 16Q212L. This level of JNK activation was similar to that achieved with nerve growth factor, a strong inducer of PC12 cell differentiation. Supportive of a role for JNK activation in PC12 cell differentiation, retrovirus-mediated overexpression of cJun, a JNK target, in PC12 cells induced neurite outgrowth. The results define a p42/44 MAP kinase-independent mechanism for differentiation of PC12 cells and suggest that persistent activation of the JNK members of the proline-directed protein kinase family by GTPase-deficient G alpha q and G alpha 16 subunits is sufficient to induce differentiation of PC12 cells.

403 Characterization of mAb AP422, a novel phosphorylation-dependent monoclonal antibody against Tau protein

Modified forms of tau proteins are major components of the paired helical filaments (PHFs) present in Alzheimer brains. In this study, tau from cytosolic samples obtained from normal and Alzheimer disease brains were fractionated by iron-chelated affinity chromatography (ICAC) to discriminate between isoforms phosphorylated to different extents using an stepwise pH gradient. Immunoblot analysis of the different fractions using antibody Tau-1 (recognizing art unphosphorylated epitope in tau and in PHF-tau after dephosphorylation) and antibody SMI 31 (recognizing a phosphorylated epitope in PHF-tau) have been carried out. Phosphorylated tau species (Tau 1-nonreactive and SMI 31-reactive) are only isolated from the Alzheimer samples at pH = 8.5. These tau species although having other Ser/Thr-Pro motifs susceptible of phosphorylation by proline-directed protein kinases are not further phosphorylated in vitro by MAP2 kinase whereas the fraction isolated at pH 7.0, which contains underphosphorylated tau species, is phosphorylated. Thus, soluble tau species phosphorylated both at the sites constituting the Tau-1 and the SMI 31 epitopes are present in Alzheimer but not in normal brain cytosol and can be isolated by ICAC. These modifications may be a prerequisite for PHF formation.

Characterization of microtubule-associated protein MAP1B: phosphorylation state, light chains, and binding to microtubules.

We have recently described a procedure for the purification of microtubule associated protein 1B (MAP1B) from calf brain [Pedrotti, B., & Islam K. (1995) Cell Motil. Cytoskeleton 30, 301-309], and this study further characterizes the purified protein and its interaction with microtubules. We show that purified MAP1B (1) is thermostable; (2) is mainly phosphorylated at the casein kinase II (CKII) sites but only partially phosphorylated at the proline-directed protein kinase (PDPK) sites; (3) both the CKII and PDPK sites can be dephosphorylated by alkaline phosphatase; and (4) dephosphorylation results in an increased mobility on SDS-PAGE gels. The ability of MAP1B to interact with microtubules was also examined and shows that (1) phosphorylated (1B-P), alkaline phosphatase-treated (1B-AP), and heat-treated (1B-P), alkaline phosphatase-treated (1B-AP), and heat-treated (1B-HT) MAP1B bind to taxol-stabilized microtubules; (2) 1 mol of 1B-P, 1B-AP, or 1B-HT each binds about 13-14 tubulin dimers; (3) light chain interaction with MAP1B heavy chain is not affected by AP- or heat-treatment; (4) MAP1B can be displaced from taxol-stabilized microtubules by titration with salt; (5) higher salt concentrations are required to displace 1B-AP compared with 1B-P from taxol-stabilized microtubules; and (6) MAP2 is able to displace both 1B-P and 1B-AP from taxol-stabilized microtubules. The role of phosphorylation in regulating MAP1B interaction with microtubules and light chains is discussed.

405 Tau in fibroblasts with and without the Swedish APP 670/671 mutation

Modified forms of tau proteins are major components of the paired helical filaments (PHFs) present in Alzheimer brains. In this study, tau from cytosolic samples obtained from normal and Alzheimer disease brains were fractionated by iron-chelated affinity chromatography (ICAC) to discriminate between isoforms phosphorylated to different extents using an stepwise pH gradient. Immunoblot analysis of the different fractions using antibody Tau-1 (recognizing art unphosphorylated epitope in tau and in PHF-tau after dephosphorylation) and antibody SMI 31 (recognizing a phosphorylated epitope in PHF-tau) have been carried out. Phosphorylated tau species (Tau 1-nonreactive and SMI 31-reactive) are only isolated from the Alzheimer samples at pH = 8.5. These tau species although having other Ser/Thr-Pro motifs susceptible of phosphorylation by proline-directed protein kinases are not further phosphorylated in vitro by MAP2 kinase whereas the fraction isolated at pH 7.0, which contains underphosphorylated tau species, is phosphorylated. Thus, soluble tau species phosphorylated both at the sites constituting the Tau-1 and the SMI 31 epitopes are present in Alzheimer but not in normal brain cytosol and can be isolated by ICAC. These modifications may be a prerequisite for PHF formation.

Protein Phosphatase 2A Is the Major Enzyme in Brain that Dephosphorylates τ Protein Phosphorylated by Proline‐Directed Protein Kinases or Cyclic AMP‐Dependent Protein Kinase

The paired helical filament (PHF), which makes up the major fibrous component of the neurofibrillary lesions of Alzheimer's disease, is composed of hyperphosphorylated and abnormally phosphorylated microtubule-associated protein tau. Previous studies have identified serine and threonine residues phosphorylated in PHF-tau and have shown that tau can be phosphorylated at several of these sites by proline-directed protein kinases and cyclic AMP-dependent protein kinase. Here we have investigated which protein phosphatase activities can dephosphorylate recombinant tau phosphorylated with mitogen-activated protein kinase, glycogen synthase kinase-3 beta, neuronal cdc2-like kinase, or cyclic AMP-dependent protein kinase. We show that protein phosphatase 2A is by far the major protein phosphatase activity in brain that dephosphorylates tau phosphorylated in this manner.

Association of protein kinase FA/GSK-3alpha (a proline-directed kinase and a regulator of protooncogenes) with human cervical carcinoma dedifferentiation/progression.

Computer analysis of protein phosphorylation-sites sequence revealed that most transcriptional factors and viral oncoproteins are prime targets for regulation of proline-directed protein phosphorylation, suggesting an association of proline-directed protein kinase (PDPK) family with neoplastic transformation and tumorigenesis. In this report, an immunoprecipitate activity assay of protein kinase FA/glycogen synthase kinase-3alpha (kinase FA/GSK-3alpha) (a particular member of PDPK family) has been optimized for human cervical tissue and used to demonstrate for the first time significantly increased (P < 0.001) activity in poorly differentiated cervical carcinoma (82.8 +/- 6.6 U/mg of protein), moderately differentiated carcinoma (36.2 +/- 3.4 U/mg of protein), and well-differentiated carcinoma (18.3 +/- 2.4 U/mg of protein) from 36 human cervical carcinoma samples when compared to 12 normal controls (4.9 +/- 0.6 U/mg of protein). Immunoblotting analysis further revealed that increased activity of kinase FA/GSK-3alpha in cervical carcinoma is due to overexpression of protein synthesis of the kinase. Taken together, the results provide initial evidence that overexpression of protein synthesis and cellular activity of kinase FA/GSK-3alpha may be involved in human cervical carcinoma dedifferentiation/progression, supporting an association of proline-directed protein kinase with neoplastic transformation and tumorigenesis. Since protein kinase FA/GSK-3alpha may function as a possible regulator of transcription factors/proto-oncogenes, the results further suggest that kinase FA/GSK-3alpha may play a potential role in human cervical carcinogenesis, especially in its dedifferentiation and progression.