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PubMed1995

The Ca2+ signaling capacity of the beta 2-integrin on HL60-granulocytic cells is abrogated following phosphorylation of its CD18-chain: relation to impaired protein tyrosine phosphorylation.

The phosphorylation state of the CD18-chain of beta 2-integrins have been shown not to mediate changes in the avidity of these receptors (i.e., inside-out signaling); however, no alternative functional significance has been proposed. Our study focused on how changes in the phosphorylation state of beta 2-integrin-receptors on HL60-granulocytic cells are related to its intracellular signal transduction properties (i.e., outside-in signaling). Engagement of beta 2-integrins on differentiated HL60 cells induced a transient increase in the cytosolic free Ca2+ concentration and an increased tyrosine phosphorylation of three major protein bands (70, 115, and 140 kDa). These signaling events occurred without any detectable phosphorylation of the CD18-chain. However, a strong phosphorylation of the CD18-chain by preexposure to phorbol myristate acetate (PMA) coincided with an abolishment of both the beta 2-integrin-induced Ca2+ signal and the protein tyrosine phosphorylations. By comparison, none of these effects were exhibited by 4-alpha-PMA, an analogue that does not activate protein kinase C. Thus, phosphorylation of the CD18-chain of beta 2-integrins is not required for outside-in signal transduction by these receptors, but it could constitute an effective mechanism by which the signaling properties of beta 2-integrins can be modulated by exogenous factors and possibly also by intracellular signals induced by other receptors. The fact that both the cytosolic free Ca2+ signal and protein tyrosine phosphorylations were abrogated by PMA suggests an intimate relationship between these two intracellular signals. To explore this possible relationship, we chelated the beta 2-integrin-induced Ca2+ signal with BAPTA. The beta 2-integrin-induced protein tyrosine phosphorylations were blocked by BAPTA but not by abolishment of the Ca2+ signal due to chelation with MAPT or by pretreatment with thapsigargin. These findings and the observation that pretreatment of cells with methyl-2,5-dihydroxycinnamate (a tyrosine kinase inhibitor) blocked the beta 2-integrin- but not the fMet-Leu-Phe-induced Ca2+ signal suggest that beta 2-integrin-induced tyrosine kinase activation occurs prior to and is a prerequisite for the subsequent Ca2+ signal.

CD18 Antigens↗
PubMed1996

Protein phosphorylation in human peripheral nerve: altered phosphorylation of a 25-kDa glycoprotein in leprosy.

Protein phosphorylation in a low speed supernatant of human peripheral nerve (tibial and sural) homogenate was investigated. The major phosphorylated proteins had molecular mass in the range of 70, 55, 45, and 25 kDa. Mg2+ or Mn2+ was essential for maximum phosphorylation although Zn2+, Co2+, and Ca2+ could partially support phosphorylation. External protein substrates casein and histone were also phosphorylated. The protein phosphatase inhibitor orthovanadate enhanced the phosphorylation of the 45 and 25 kDa proteins significantly. Concanavalin A-Sepharose chromatography of the phosphorylated peripheral nerve proteins showed that the 25 kDa protein was a glycoprotein. Protein phosphorylation of peripheral nerves from leprosy affected individuals was compared with normals. The phosphorylation of 25 kDa protein was decreased in most of the patients with leprosy.

Case-Control Studies↗
PubMed1985

Effect of sulfhydryl-disulfide state on protein phosphorylation: phosphorylation of bovine serum albumin.

Bovine serum albumin (BSA) was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase under general protein phosphorylation conditions. The optimal pH for this phosphorylation was 9.0. The K0.5 (the concentration required for 50% of maximal phosphorylation) for BSA at pH 7.5 was 15 microM. One mole of phosphate was incorporated per mole of BSA, and only one phosphopeptide fragment was obtained after extensive proteolysis with trypsin. BSA phosphorylation required dithiothreitol or GSH, but GSH was only one-fiftieth as effective as dithiothreitol. GSSG counteracted the effect of dithiothreitol and GSH. Phosphorylation increased in a time-dependent and dithiothreitol concentration-dependent manner when BSA was preincubated with dithiothreitol. The increase in the incorporation of 32P correlated with the appearance of up to six free sulfhydryl groups. The effect of dithiothreitol on BSA appeared reversible, since reoxidation of reduced BSA decreased its susceptibility to phosphorylation. These experiments showed that this in vitro phosphorylation is dependent on the sulfhydryl-disulfide state of BSA. The possible implications of the sulfhydryl-disulfide state of proteins in the regulation of phosphorylation are discussed.

Animals↗
PubMed1991

Comparison of insulin receptor tyrosine phosphorylation under in vitro and in situ conditions: assessment of specific protein tyrosine phosphorylation without the use of 32P-phosphate-labeled substrates.

An assay which permitted a comparison of insulin receptor tyrosine phosphorylation induced under in vivo and in situ conditions was developed. It was demonstrated that the level of tyrosine phosphorylation of the receptor induced by insulin under in situ conditions exceeded by 2.3-fold the level of tyrosine phosphorylation induced under in vitro conditions. In addition, chronically treated, down-regulated cells demonstrated a significant decrease in the level of insulin receptor tyrosine phosphorylation compared to cells acutely treated with insulin. The procedure utilized an antibody specific for the insulin receptor which allowed separation of the receptor from other cellular proteins which are potential substrates for tyrosine phosphorylation. A second iodinated antibody specifically directed against phosphotyrosine residues allowed quantitation of the phosphorylated residues. Since the use of 32P-labeled substrates for phosphorylation was not required, this procedure allowed a comparison of protein phosphorylation induced under in vitro and in situ conditions. This procedure should permit investigations into the roles of other cellular factors, such as phosphatases and serine kinases, in modulating protein tyrosine phosphorylation.

Antibodies↗
PubMed1996

Quantitative image analysis of temporal changes in tau and neurofilament proteins during the course of acute experimental neurofibrillary degeneration; non-phosphorylated epitopes precede phosphorylation.

Perturbation of the neuronal cytoskeleton represents an integral feature of neurofibrillary tangles which are characteristic neuropathological findings seen in Alzheimer's disease. Microtubule associated protein tau (tau) is considered to be the major component of these lesions although neurofilament proteins also are present. The present study explores the formation of intraneuronal tau and neurofilament protein aggregates using intracisternal administration of aluminum maltolate to rabbits. The time course of the formation of these aggregates and subsequent phosphorylation have been investigated by immunohistochemical methods using a panel of monoclonal antibodies, with quantitation of the staining by image analysis. Neurofilament proteins begin to aggregate by day 1 following aluminum maltolate injection on day 0. Increases in non-phosphorylated neurofilament proteins are observed first, with phosphorylated epitopes being recognized by day 3. Tau follows a similar pattern in that non-phosphorylated epitopes appear to precede phosphorylation. The monoclonal antibody Alz-50 which recognizes a phosphorylation-independent epitope of tau in Alzheimer's disease paired helical filaments, demonstrates positivity in the aluminum maltolate-treated rabbits by day 3. Other tau monoclonal antibodies which recognize phosphorylated tau in paired helical filaments (AT8 and PHF-1) show positive immunostaining on days 6-8. These results indicate that intraneuronal aggregation of cytoskeletal proteins can be initiated by factors other than phosphorylation. However, phosphorylation occurring as a secondary event probably contributes to stabilization of the aggregates.

Animals↗
PubMed1988

Phosphorylation and dephosphorylation of hormone-sensitive lipase. Interactions between the regulatory and basal phosphorylation sites.

Phosphorylation of the basal site with glycogen synthase kinase-4 enhanced the rate of phosphorylation of the regulatory site by cyclic AMP-dependent protein kinase 1.7-fold. In contrast, the phosphorylation state of the regulatory site did not affect the rate of phosphorylation of the basal site with glycogen synthase kinase-4. The rate of dephosphorylation of either the regulatory or the basal phosphorylation site by protein phosphatase-1, 2A or 2C was independent of the phosphorylation state of the other site. These results suggest that the basal phosphorylation site could play an indirect role in the control of the hormone-sensitive lipase activity in the adipocyte by functioning as a recognition site for the cyclic AMP-dependent protein kinase in the phosphorylation of the activity-controlling regulatory phosphorylation site in response to lipolytic hormones.

Animals↗
PubMed2004

Activation of formyl peptide receptor-like 1 by WKYMVm induces serine phosphorylation of STAT3, which inhibits its tyrosine phosphorylation and nuclear translocation induced by hydrogen peroxide.

Signal transducer and activator of transcription 3 (STAT3) is an important transcription factor that modulates the expression of several genes. The activation of STAT3 accompanies tyrosine phosphorylation and its translocation to the nucleus. Formyl peptide receptor like 1 (FPRL1) is an important classical chemoattractant receptor. In this study, we observed that the stimulation of FPRL1 by Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm) caused serine phosphorylation but not tyrosine phosphorylation of STAT3 in a pertussis toxin-sensitive manner. Moreover, downstream of FPRL1 stimulation, phospholipase D (PLD) activity was dramatically increased. n-butanol, a well-known phosphatidic acid (PA) acceptor, completely inhibited WKYMVm-induced STAT3 serine phosphorylation. Moreover, the exogenous addition of PA mimicked STAT3 phosphorylation by WKYMVm. We also found that WKYMVm stimulated extracellular signal regulated kinase (ERK), and that ERK activity is required for STAT3 serine phosphorylation. This WKYMVm-induced ERK activation was inhibited by n-butanol, whereas ERK activation was also induced by the addition of exogenous PA. In terms of the functional aspects of the WKYMVm-induced serine phosphorylation of STAT3, we found that hydrogen peroxide-stimulated STAT3 activation was blocked by pretreating WKYMVm. Taken together, we found that WKYMVm stimulated FPRL1, and that this resulted in STAT3 serine phosphorylation via PLD-mediated ERK activation, and that the serine phosphorylation of STAT3 blocked hydrogen peroxide-induced STAT3 activity.

Biotransformation↗
PubMed2005

Site-specific phosphorylation of SCG10 in neuronal plasticity: role of Ser73 phosphorylation by N-methyl D-aspartic acid receptor activation in rat hippocampus.

Accumulated evidence suggests that actin and microtubule regulating proteins contribute to neuronal structural dynamics, which subsequently affect neuronal plasticity. SCG10 is a neuronal-specific stathmin protein with microtubule destabilizing activity that is affected by multiple phosphorylation, at least in vitro. SCG10 has four major phosphorylation sites: Ser50 and Ser97 targeted by protein kinase A (PKA), and Ser62 and Ser73 targeted by mitogen-activated protein kinase (MAPK). To explore the potential roles of site-specific phosphorylation in physiological models, we developed phosphorylation site-specific antibodies and examined the SCG10 status in primary cultured hippocampal neurons and tissues. Although SCG10 is concentrated in growth cones and the Golgi apparatus in primary cultured neurons, the phosphorylated form was also detected in both regions, suggesting that MT dynamics within the growth cone may be regulated by protein phosphorylation. In the adult hippocampus, an intense stimulus such as kainate treatment induced a rapid phosphorylation of Ser73 within 15 min that was sustained for at least 60 min. This response was mediated through the N-methyl D-aspartic acid (NMDA) receptor and was ablated by the antagonist MK-801. The MAPK enzyme Erk2 was simultaneously activated along a similar time course to SCG10, suggesting that Erk2 may directly phosphorylate Ser73. These results demonstrate that changes in the phosphorylation status of SCG10 in vivo, dependent upon neural activity and/or plasticity, could affect the microtubule dynamics in neuronal dendrites.

Amino Acid Sequence↗
PubMed1998

Tyrosine phosphorylation of proteins in primary human myeloid leukemia cells stimulated by cytokines: analysis of the frequency of phosphorylation, and partial identification and semi-quantification of signaling molecules.

We investigated tyrosine phosphorylation of proteins in primary human leukemia cells stimulated by granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), interleukin-3 (IL-3), tumor necrosis factor (TNF), thrombopoietin (TPO) and phorbol myristate acetate (PMA) in 61 patients with acute myeloid leukemia (AML), nine patients with chronic myeloid leukemia (CML) in blastic crisis and four patients in chronic phase, and compared these data of leukemia with those of normal human immature hematopoietic cells. These cytokines and PMA induced tyrosine phosphorylation of proteins in a manner characteristic for each cytokine or PMA in AML cells. G-CSF, GM-CSF and IL-3 frequently phosphorylated p92, p80, p70, p44 and p42. p95 was frequently phosphorylated by G-CSF, and was phosphorylated in one third of the cases by TPO. On the other hand, TNF selectively induced tyrosine phosphorylation of p42, and PMA selectively induced that of p44 and p42. In marked contrast to AML cells, CML cells responded poorly to cytokines with protein tyrosine phosphorylation, and normal human bone marrow mononuclear cells and CD34-positive cells also showed poor response to cytokines. The results of the immunoprecipitation studies showed tyrosine phosphorylation of signal transducers and activators of transcription (Stat) 5 induced by G-CSF, GM-CSF, IL-3 and/or TPO in six cases, that of extracellular signal-regulated kinase (ERK) by GM-CSF in two cases and that of p38 by TNF in three cases. Intracellular amount of Stat5 was markedly increased in AML cells compared with that in CML cells and normal human bone marrow cells. whereas intracellular amount of ERK and p38 was uniformly abundant in both leukemic and normal cells. These results show cytokine-specific and amplified tyrosine phosphorylation of proteins in AML cells and suggest that amplified response might, at least in part, result from the increased amount of signaling molecules such as Stat5.

Acute Disease↗
PubMedDate not supplied

Connexin 36 in bovine retina: lack of phosphorylation but evidence for association with phosphorylated proteins.

In vertebrate retina interneuronal communication through gap junctions is involved in light adaptation and in the transfer of visual information from the rod pathway to the cone pathway. Reports over the last two decades have indicated that these gap junctions are regulated by cyclic nucleotide-dependent protein kinases suggesting that the gap junction proteins, connexins, are phosphorylated. Though all the connexins involved in light adaptation and information transfer from rod to cone pathway are not yet known, connexin 36 has been shown to be definitively involved in the latter process. We have therefore attempted to investigate the cyclic nucleotide-dependent phosphorylation of this connexin in bovine retina. We found several soluble and membrane proteins in bovine retina whose phosphorylation was regulated by cyclic nucleotides. However, no protein of about 36 kDa with cyclic nucleotide-regulated phosphorylation was found in gap junction-enriched membrane preparations. A 36-kDa phosphorylated protein was found in gap junction-enriched membranes phosphorylated in the presence of calcium. However, this protein was not immunoprecipitated by anti-connexin 36 antibodies indicating that it was not connexin 36 in spite of its similarity in molecular weight. Immunoprecipitation did reveal phosphorylated proteins coimmunoprecipitated with connexin 36. Two of these proteins were identified as beta and alpha tubulin subunits. Though cyclic GMP and calcium did not greatly influence the association of these proteins with connexin 36, the results suggest the possibility of connexin 36 associating with other proteins. Together, these observations indicate that interneuronal communication at gap junctions made by connexin 36 may not be regulated by direct phosphorylation of connexin 36, but possibly by phosphorylation of associated proteins.

Animals↗
PubMed1984

Phosphorylation kinetics of skeletal muscle myosin and the effect of phosphorylation on actomyosin adenosinetriphosphatase activity.

Purified rabbit skeletal muscle myosin is phosphorylated on one type of light-chain subunit (P-light chain) by calmodulin-dependent myosin light chain kinase and dephosphorylated by phosphoprotein phosphatase C. Analyses of the time courses of both phosphorylation and dephosphorylation of skeletal muscle myosin indicated that both reactions, involving at least 90% of the P-light chain, were kinetically homogeneous. These results suggest that phosphorylation and dephosphorylation of rabbit skeletal muscle myosin heads are simple random processes in contrast to the sequential phosphorylation mechanism proposed for myosin from gizzard smooth muscle. We also examined the effect of phosphorylation of rabbit skeletal muscle myosin on the actin-activated ATPase activity. We observed an apparent 2-fold decrease in the Km for actin, from about 6 microM to about 2.5 microM, with no significant effect on the Vmax (1.8s-1) in response to P-light-chain phosphorylation. There was no significant effect of phosphorylation on the ATPase activity of myosin alone (0.045 s-1). ATPase activation could be fully reversed by addition of phosphatase catalytic subunit. The relationship between the extents of P-light-chain phosphorylation and ATPase activation (at 3.5 microM actin and 0.6 microM myosin) was essentially linear. Thus, in contrast to results obtained with myosin from gizzard smooth muscle, these results suggest that cooperative interactions between the myosin heads do not play an important role in the activation process in skeletal muscle. Since the effect of P-light-chain phosphorylation is upon the Km for actin, it would appear to be associated with a significant activation of ATPase activity only at appropriate concentrations of actin and salt.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphatases↗
PubMed1996

Phosphorylation of C8 and C9 subunits of the multicatalytic proteinase by casein kinase II and identification of the C8 phosphorylation sites by direct mutagenesis.

Two 29 kDa subunits of the multicatalytic proteinase (proteasome) complex, the C8 and C9 components, are phosphorylated in vivo and can be phosphorylated in vitro by casein kinase II (CKII). The major phosphate acceptor is the C8 subunit being phosphorylated in serine, both in vivo and in vitro. The phosphopeptides generated by Glu-C endoprotease digestion from the in vivo 29 kDa labeled subunit and from the in vitro phosphorylation of the recombinant C8 subunit with CKII are identical, suggesting that CKII is likely responsible for the in vivo phosphorylation of the C8 subunit. The in vitro stoichiometry of phosphorylation of the proteasome complex and the recombinant C9 and C8 subunits by CKII is 2-2.5, 0.2, and 2 mol of phosphate per mole, respectively. Several C8 protein constructs allow the location of the CKII phosphorylation sites to be the COOH terminal portion of the protein, and direct mutational analyses show that Ser-243 and Ser-250 are the residues of the C8 subunit phosphorylated by CKII. The in vitro phosphorylation of the proteasome by CKII does not affect its proteolytic activity (on proteins or fluorogenic synthetic peptides), therefore suggesting its involvement in the interaction of the proteasome with other cellular proteins, i.e. in the formation of the 26S complex and/or in the interaction with the nuclear translocation machinery.

Amino Acid Sequence↗
PubMed2004

Phosphorylation regulates nucleophosmin targeting to the centrosome during mitosis as detected by cross-reactive phosphorylation-specific MKK1/MKK2 antibodies.

Phosphorylation-specific antibodies provide a powerful tool for analysing the regulation and activity of proteins in the MAP (mitogen-activated protein) kinase and other signalling pathways. Using synchronized cells, it was observed that phosphorylation-specific antibodies developed against the active form of MKK1/MKK2 (MAP kinase kinase-1 and -2) reacted with a protein that was approx. 35 kDa during G2/M-phase of the cell cycle. Failure of the 35 kDa protein to react with phosphorylation-independent MKK1/MKK2 antibodies suggested that this protein was not related to MKK1 or MKK2. Thus the 35 kDa protein was isolated by immunoprecipitation with the phospho-MKK1/MKK2 antibody and identified by MS. Peptide sequence analysis revealed matches with NPM (nucleophosmin/B23), a phosphoprotein involved in nucleolar assembly, centrosome duplication and ribosome assembly and transport. Biochemical and immunocytochemistry analyses verified that the phospho-MKK1/MKK2 antibodies cross-reacted with NPM that was phosphorylated at Thr234 and Thr237 during G2/M-phase, which are the same sites that are targeted by Cdc2 (cell division cycle protein-2) during mitosis. Using phosphorylation site mutants, we show that phosphorylation of Thr234 and Thr237 is required for NPM immunoreactivity with the phospho-MKK1/MKK2 antibody. Moreover, phosphorylation of Thr234 and Thr237 was demonstrated to regulate NPM localization to the centrosome after nuclear envelope breakdown in mitotic cells. These findings reveal a new insight into the role of phosphorylation in regulating NPM targeting during mitosis. However, caution should be used when using commercially available phospho-MKK1/MKK2 antibodies to examine the regulation of MKK1/MKK2 during mitotic transitions, owing to their cross-reactivity with phosphorylated NPM at this time of the cell cycle.

Animals↗
PubMed1997

Phosphorylation of complement component C3 after synthesis in U937 cells by a putative protein kinase, casein kinase 2, which is regulated by CD11b: evidence that membrane-bound proteases preferentially cleave phosphorylated C3.

It was our aim in this study to investigate the possibility that the third component of complement (C3) is phosphorylated during synthesis and secretion in U937 cells. Labelling of U937 cells with [32P]Pi, followed by immunoprecipitation of C3 from cell lysates and culture supernatants at different time points, showed that C3 was phosphorylated intracellularly immediately before release into the medium, which initiated cleavage of the protein into an iC3b-like fragment. Stimulation of CD11b/CD18 increased phosphorylation 7-fold, from a basal level of 2%. The phosphorylation sites in C3 did not resemble those described previously for casein kinase (CK) 1, cAMP-dependent protein kinase A or calcium- and phospholipid-dependent protein kinase C. Instead, protein kinase CK2 was suggested inasmuch as: (1) CK2 was detected both on the cell surface and on shed microparticles; (2) phosphorylation of purified C3 by microparticles was abolished by a monoclonal antibody, anti-CK2; (3) the [32P]Pi tag of both phosphorylated C3 (secreted from U937 cells) and of microparticle-phosphorylated C3, which was cleaved either by membrane proteases or by leucocyte elastase, was found in a 40 and a 70 kDa polypeptide; (4) both secreted C3 and C3 phosphorylated in vitro were much more susceptible to cleavage by proteases. Generation of C3 fragments provides a means by which U937 cells can stimulate nearby cells which are expressing complement receptors. The present study demonstrates that the cleavage of C3 is controlled by an intracellular phosphorylation event regulated by CD11b/CD18.

Casein Kinase II↗
PubMed1998

GTP-binding protein gamma12 subunit phosphorylation by protein kinase C--identification of the phosphorylation site and factors involved in cultured cells and rat tissues in vivo.

We have demonstrated previously that the GTP-binding protein gamma12 subunit is a selective substrate for phosphorylation by protein kinase C among various gamma subunits in vitro, and that a serine residue in the N-terminal region is involved. In the present study, we first determined that the site of phosphorylation was Ser1 with antibodies developed against two N-terminal peptides containing phosphorylated Ser1 and Ser2, respectively. Using an antibody recognizing phosphorylated gamma12 and Swiss 3T3 cells rich in this protein, gamma12 was found to be phosphorylated by stimulation of quiescent cells with various reagents, such as phorbol 12-myristate 13-acetate (PMA), NaF, fetal calf serum, lysophosphatidic acid, endothelin, and growth factors. Pertussis toxin completely and partially prevented phosphorylation of gamma12 induced by lysophosphatidic acid and fetal calf serum and by endothelin, respectively, suggesting a contribution of G(i/o). Phosphorylation of gamma12 was limited when cells were stimulated by a single reagent, even with PMA, a strong activator of protein kinase C, whereas simultaneous stimulation with lysophosphatidic acid and either PMA or platelet-derived growth factor induced a synergistic increase of phosphorylation, suggesting physiological roles for GTP-binding proteins and protein kinase C in combination. Phosphorylated gamma12 was also detected in various tissues of untreated rats. Its decrease by pertussis toxin treatment also suggested the involvement of G(i/o) in vivo.

3T3 Cells↗
PubMed1994

Tau phosphorylation in human, primate, and rat brain: evidence that a pool of tau is highly phosphorylated in vivo and is rapidly dephosphorylated in vitro.

The extent of tau phosphorylation is thought to regulate the binding of tau to microtubules: Highly phosphorylated tau does not bind to tubules, whereas dephosphorylated tau can bind to microtubules. It is interesting that the extent of tau phosphorylation in vivo has not been accurately determined. Tau was rapidly isolated from human temporal neocortex and hippocampus, rhesus monkey temporal neocortex, and rat temporal neocortex and hippocampus under conditions that minimized dephosphorylation. In brain slices, we observed that tau isolated under such conditions largely existed in several phosphorylated states, including a pool that was highly phosphorylated; this was determined using epitope-specific monoclonal and polyclonal antibodies. This highly phosphorylated tau was dephosphorylated during a 120-min time course in vitro, presumably as a result of neuronal phosphatase activity. The slow-mobility forms of tau were shifted to faster-mobility forms following in vitro incubation with alkaline phosphatase. Laser densitometry was used to estimate the percent of tau in slow-mobility, highly phosphorylated forms. Approximately 25% of immunoreactive tau was present as slow-mobility (66- and 68-kDa) forms of tau. The percentage of immunoreactive tau in faster-mobility pools (42-54 kDa) increased in proportion to the decrease in content of 66-68-kDa tau as a function of neuronal phosphatases or alkaline phosphatase treatment. These data suggest that the turnover of phosphorylated sites on tau is rapid and depends on neuronal phosphatases. Furthermore, tau is highly phosphorylated in normal-appearing human, primate, and rodent brain.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed1996

Studies on the in vitro phosphorylation of HSSB-p34 and -p107 by cyclin-dependent kinases. Cyclin-substrate interactions dictate the efficiency of phosphorylation.

Cyclin-dependent kinases (Cdks) are required for cell cycle progression. Two potentially significant Cdk substrates in human cells are the human single-stranded binding protein (HSSB or RPA), which plays an essential role in DNA replication, repair, and recombination, and the tumor suppressor p107 which acts to negatively regulate cell growth. In this report we describe the in vitro phosphorylation of these two proteins by Cdks in an attempt to understand how cyclin-substrate interactions direct phosphorylation efficiencies. We show that cyclin A-Cdk2 efficiently phosphorylates the p34 subunit of HSSB (HSSB-p34) alone or as a part of the heterotrimeric complex. In contrast, cyclin E-Cdk2 that is active in phosphorylating histone H1, does not support the phosphorylation of the p34 subunit of HSSB. We provide evidence that this differential phosphorylation results from a specific interaction between HSSB-p34 and cyclin A, but not cyclin E. Thus the observed cell cycle-dependent phosphorylation of HSSB-p34 at the G1 to S transition is most likely catalyzed by cyclin A-Cdk2 initiated by the direct interaction between cyclin A and the HSSB-p34 subunit. These studies are consistent with our previous observation that p107, which directly binds cyclin A, is efficiently phosphorylated by cyclin A-Cdk2 but not cyclin B-associated kinases. Here we further demonstrate that cyclin A only complexes with p107 in its unphosphorylated form. These data suggest a catalytic mechanism by which Cdk acts: substrate targeting by a cyclin-substrate interaction followed by dissociation of the Cdk upon phosphate incorporation allowing the Cdk to become available for the next cycle of phosphorylation.

Base Sequence↗
PubMed1997

Tyrosine phosphorylation of Crk-associated substrates by focal adhesion kinase. A putative mechanism for the integrin-mediated tyrosine phosphorylation of Crk-associated substrates.

Integrin-ligand binding induces the tyrosine phosphorylation of various proteins including focal adhesion kinase (pp125(FAK)) and Crk-associated substrate (Cas). FAK is activated and autophosphorylated by the ligation of integrins, although the substrate of FAK has not been revealed. We show here that p130(Cas) and Cas-L are FAK substrates. FAK directly phosphorylates Cas proteins primarily at the YDYVHL sequence that is conserved among all Cas proteins. Furthermore, the phosphorylated YDYVHL sequence is a binding site for Src family protein-tyrosine kinases, and the recruited Src family kinase phosphorylates the other tyrosine residues within Cas. The Cas-L YDYVHL sequence is phosphorylated upon integrin-ligand binding, and this integrin-mediated tyrosine phosphorylation is inhibited by the cotransfection of the FAK COOH-terminal domain that does not contain a kinase domain. These findings strongly suggest that FAK initiates integrin-mediated tyrosine phosphorylation of Cas proteins; then, Src family tyrosine kinases, which are recruited to phosphorylated Cas and FAK, further phosphorylate Cas proteins.

Adaptor Proteins, Signal Transducing↗