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R B Pearson

Publications and source records attributed to R B Pearson.

At least 19 recordsLinked to original sources
PubMed2003

Protein kinase-dependent phosphorylation of the Menkes copper P-type ATPase.

The Menkes copper-translocating P-type ATPase (ATP7A; MNK) is a key regulator of copper homeostasis in humans. It has a dual role in supplying copper to essential cuproenzymes in the trans-Golgi network (TGN) and effluxing copper from the cell. These functions are achieved through copper-regulated trafficking of MNK between the TGN and the plasma membrane. However, the exact mechanism(s) which regulate the localisation and biochemical functions of MNK are still unknown. Here we investigated copper-dependent phosphorylation of MNK by a putative protein kinase(s). We found that in the presence of elevated copper there was a substantial increase in phosphorylation of the wild-type MNK in vivo. The majority of copper-dependent phosphorylation was on serine residues in two phosphopeptides. In contrast, there was no up-regulation of phosphorylation of a non-trafficking MNK mutant with mutated cytosolic copper-binding sites. Our findings suggest a potentially important role of kinase-dependent phosphorylation in the regulation of function of the MNK protein.

Adenosine Triphosphatases↗
PubMed2001

Phosphorylation of nuclear phospholipase C beta1 by extracellular signal-regulated kinase mediates the mitogenic action of insulin-like growth factor I.

It is well established that a phosphoinositide (PI) cycle which is operationally distinct from the classical plasma membrane PI cycle exists within the nucleus, where it is involved in both cell proliferation and differentiation. However, little is known about the regulation of the nuclear PI cycle. Here, we report that nucleus-localized phospholipase C (PLC) beta1, the key enzyme for the initiation of this cycle, is a physiological target of extracellular signal-regulated kinase (ERK). Stimulation of Swiss 3T3 cells with insulin-like growth factor I (IGF-I) caused rapid nuclear translocation of activated ERK and concurrently induced phosphorylation of nuclear PLC beta1, which was completely blocked by the MEK inhibitor PD 98059. Coimmunoprecipitation detected a specific association between the activated ERK and PLC beta1 within the nucleus. In vitro studies revealed that recombinant PLC beta1 could be efficiently phosphorylated by activated mitogen-activated protein kinase but not by PKA. The ERK phosphorylation site was mapped to serine 982, which lies within a PSSP motif located in the characteristic carboxy-terminal tail of PLC beta1. In cells overexpressing a PLC beta1 mutant in which serine 982 is replaced by glycine (S982G), IGF-I failed to activate the nuclear PI cycle, and its mitogenic effect was also markedly attenuated. Expression of S982G was found to inhibit ERK-mediated phosphorylation of endogenous PLC beta1. This result suggests that ERK-evoked phosphorylation of PLC beta1 at serine 982 plays a critical role in the activation of the nuclear PI cycle and is also crucial to the mitogenic action of IGF-I.

3T3 Cells↗
PubMed2000

STIM1: a novel phosphoprotein located at the cell surface.

STIM1 is a novel candidate growth suppressor gene mapping to the human chromosome region 11p15.5 that is associated with several malignancies. STIM1 overexpression studies in G401 rhabdoid tumour, rhabdomyosarcoma and rodent myoblast cell lines causes growth arrest, consistent with a potential role as a tumour growth suppressor. We used highly specific antibodies to show by immunofluorescence and cell surface biotinylation studies that STIM1 is located at the cell surface of K562 cells. Western blot analysis revealed that the 90-kDa STIM1 protein is ubiquitously expressed in various human primary cells and tumour cell lines. STIM1 is not secreted from cells and does not appear to undergo proteolytic processing. We show evidence of post-translational modification of STIM1, namely phosphorylation and N-linked glycosylation. Phosphorylation of STIM1 in vivo occurs predominantly on serine residues. Thus, STIM1, the putative tumour growth suppressor gene is ubiquitously expressed and has features of a regulatory cell-surface phosphoprotein.

Amino Acid Sequence↗
PubMed1999

Insulin and insulin antagonists evoke phosphorylation of P20 at serine 157 and serine 16 respectively in rat skeletal muscle.

We show here that insulin and insulin antagonists differentially modify phosphorylation of three phospho-isoforms of P20 (termed S1, S2 and S3) in rat skeletal muscle. Precise phosphorylation sites of S1 and S2 were mapped to serine 157 and serine 16 respectively. Insulin evoked phosphorylation of P20 at serine 157 through the phosphatidylinositol (PI) 3-kinase pathway. Epinephrine and calcitonin gene-related peptide decreased phosphorylation at serine 157 and increased phosphorylation at serine 16 and other unidentified sites. These results demonstrate that the PI-3-kinase pathway of anabolic insulin and the cAMP pathway of catabolic hormones converge on P20 and suggest a potential role of this protein in regulating energy metabolism of skeletal muscle.

Amino Acid Sequence↗
PubMed1999

Dual posttranscriptional targets of retinoic acid-induced gene expression.

Retinoic acid-induced differentiation of the pre-osteoblastic cell line, UMR 201, is associated with a marked increase in the proficiency of posttranscriptional nuclear processing of alkaline phosphatase mRNA. In this study we attempted to correlate the posttranscriptional actions of retinoic acid with changes in phosphorylation, or abundance of spliceosome components, or both. Treatment with retinoic acid for periods of < or = 4 h resulted in dephosphorylation of nuclear U1 70K protein without affecting its abundance. Peptide mapping showed that U1 70K dephosphorylation was related to the disappearance of one specific phosphopeptide out of four major U1 70K phosphopeptides. A twofold decrease in mRNA expression of an isoform of alternative splicing factor that inhibits splicing was also observed over the same period. Tumor necrosis factor-alpha, which enhances the posttranscriptional action of retinoic acid, reduced U1 70K mRNA expression, while an inhibition of retinoic acid action by transforming growth factor-beta was associated with a marked increase in U1 70K mRNA levels. Our results draw attention to the complex interactions between short- and long-term alterations in the abundance and functional status of U1 70K, as well as SR proteins by growth and/or differentiation factors in the regulation of spliceosome formation and function.

Alkaline Phosphatase↗
PubMed1998

Phosphorylation sites in the autoinhibitory domain participate in p70(s6k) activation loop phosphorylation.

Here we have employed p70(s6k) truncation and point mutants to elucidate the role played by the carboxyl-terminal autoinhibitory domain S/TP phosphorylation sites in kinase activation. Earlier studies showed that truncation of the p70(s6k) amino terminus severely impaired kinase activation but that this effect was reversed by deleting the carboxyl terminus, which in parallel led to deregulation of Thr229 phosphorylation in the activation loop (Dennis, P. B., Pullen, N., Kozma, S. C., and Thomas, G. (1996) Mol. Cell. Biol. 16, 6242-6251). In this study, substitution of acidic residues for the four autoinhibitory domain S/TP sites mimics the carboxyl-terminal deletion largely by rescuing kinase activation caused by the amino-terminal truncation. However, these mutations do not deregulate Thr229 phosphorylation, suggesting the involvement of another regulatory element in the intact kinase. This element appears to be Thr389 phosphorylation, because substitution of an acidic residue at this position in the p70(s6k) variant containing the S/TP mutations leads to a large increase in basal Thr229 phosphorylation and kinase activity. In contrast, an alanine substitution at Thr389 blocks both responses. Consistent with these data, we show that a mutant harboring the acidic S/TP and Thr389 substitutions is an excellent in vitro substrate for the newly identified Thr229 kinase, phosphoinositide-dependent kinase-1 (Pullen, N., Dennis, P. B., Andjelkovic, M., Dufner, A., Kozma, S., Hemmings, B. A., and Thomas, G. (1998) Science 279, 707-710), whereas phosphoinositide-dependent kinase-1 poorly utilizes the two p70(s6k) variants that have only one set of mutations. These findings indicate that phosphorylation of the S/TP sites, in cooperation with Thr389 phosphorylation, controls Thr229 phosphorylation through an intrasteric mechanism.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed1998

Differential regulation by calcium reveals distinct signaling requirements for the activation of Akt and p70S6k.

Activation of the phosphatidylinositol 3-kinase (PI3K) plays an important role in the mitogenic response of many cell types. Recently, two serine/threonine kinases Akt and p70(S6k) have been identified as physiological targets of PI3K. Observations that expression of activated forms of Akt led to the activation of p70(S6k) implied Akt might mediate mitogenic signaling through activation of p70(S6k). To clarify the relationship between signaling through these two kinases, we have examined their regulation by various mitogenic stimuli. In this study we have focused on the role of calcium in the regulation of each kinase in Balb/c-3T3 fibroblasts. Depletion of intracellular calcium stores by EGTA pretreatment has no effect on growth factor-induced Akt activation but completely abolishes p70(S6k) stimulation. Increase of intracellular calcium induced by ionomycin or thapsigargin results in a full activation of p70(S6k), whereas little or no activation of Akt is observed. Furthermore, although PI3K in anti-phosphotyrosine immunoprecipitates is only very weakly activated by ionomycin, the calcium-induced stimulation of p70(S6k) is completely inhibited by the specific PI3K inhibitor wortmannin. We conclude Akt and p70(S6k) lie on separate signaling pathways. Activation of signaling to Akt is insufficient for the activation of p70(S6k), which can be achieved independently of Akt. p70(S6k) requires a separate calcium-dependent and wortmannin-sensitive process that is likely to be independent of type IA PI3K family members.

3T3 Cells↗
PubMed1998

Effects of wortmannin and rapamycin on CSF-1-mediated responses in macrophages.

There are differing views regarding the roles of phosphatidylinositol 3-kinases (PI3-kinases) and p70 S6 kinase (p70s6k) in growth factor-induced cellular responses. One approach that is widely employed to investigate these roles is to use the inhibitors, wortmannin and rapamycin, respectively. This approach is used here to study the responses in macrophages to colony stimulating factor-1 (CSF-1). Wortmannin (> or = 30 nM) and rapamycin (> or = 3 nM) both weakly inhibited CSF-1-stimulated DNA synthesis in murine bone marrow-derived macrophages (BMM), suggesting that there are PI3-kinase- and p70s6k-independent pathways required for the onset of S phase; interestingly the combination of the drugs gave dramatic suppression. Inhibition of DNA synthesis by rapamycin on the BMM was much less than that observed with the CSF-1-dependent cell line, BAC1.2F5. In BMM, wortmannin suppressed CSF-1-stimulated increase in p70s6k activity indicating that PI3-kinase activity may lie upstream. In contrast to some other growth factor/cell systems, no evidence was obtained using the inhibitors for the involvement of PI3-kinase or p70s6k in CSF-1-mediated induction of c-fos mRNA expression or Erk-1 activity; in addition, no evidence was found for an involvement in the CSF-1-mediated increase in cyclin D1 expression or STAT activation. The findings reinforce the need to study the signal transduction cascades relevant to each individual growth factor and preferably not in cell lines.

Androstadienes↗
PubMed1997

Rapamycin suppresses 5'TOP mRNA translation through inhibition of p70s6k.

Treatment of mammalian cells with the immunosuppressant rapamycin, a bacterial macrolide, selectively suppresses mitogen-induced translation of an essential class of mRNAs which contain an oligopyrimidine tract at their transcriptional start (5'TOP), most notably mRNAs encoding ribosomal proteins and elongation factors. In parallel, rapamycin blocks mitogen-induced p70 ribosomal protein S6 kinase (p70s6k) phosphorylation and activation. Utilizing chimeric mRNA constructs containing either a wild-type or disrupted 5'TOP, we demonstrate that an intact polypyrimidine tract is required for rapamycin to elicit an inhibitory effect on the translation of these transcripts. In turn, a dominant-interfering p70s6k, which selectively prevents p70s6k activation by blocking phosphorylation of the rapamycin-sensitive sites, suppresses the translation of the chimeric mRNA containing the wild-type but not the disrupted 5'TOP. Conversion of the principal rapamycin-sensitive p70s6k phosphorylation site, T389, to an acidic residue confers rapamycin resistance on the kinase and negates the inhibitory effects of the macrolide on 5'TOP mRNA translation in cells expressing this mutant. The results demonstrate that the rapamycin block of mitogen-induced 5'TOP mRNA translation is mediated through inhibition of p70s6k activation.

3T3 Cells↗
PubMed1997

Dual requirement for a newly identified phosphorylation site in p70s6k.

The activation of p70s6k is associated with multiple phosphorylations at two sets of sites. The first set, S411, S418, T421, and S424, reside within the autoinhibitory domain, and each contains a hydrophobic residue at -2 and a proline at +1. The second set of sites, T229 (in the catalytic domain) and T389 and S404 (in the linker region), are rapamycin sensitive and flanked by bulky aromatic residues. Here we describe the identification and mutational analysis of three new phosphorylation sites, T367, S371, and T447, all of which have a recognition motif similar to that of the first set of sites. A mutation of T367 or T447 to either alanine or glutamic acid had no apparent effect on p70s6k activity, whereas similar mutations of S371 abolished kinase activity. Of these three sites and their surrounding motifs, only S371 is conserved in p70s6k homologs from Drosophila melanogaster, Arabidopsis thaliana, and Saccharomyces cerevisiae, as well as many members of the protein kinase C family. Serum stimulation increased S371 phosphorylation; unlike the situation for specific members of the protein kinase C family, where the homologous site is regulated by autophosphorylation, S371 phosphorylation is regulated by an external mechanism. Phosphopeptide analysis of S371 mutants further revealed that the loss of activity in these variants was paralleled by a block in serum-induced T389 phosphorylation, a phosphorylation site previously shown to be essential for kinase activity. Nevertheless, the substitution of an acidic residue at T389, which mimics phosphorylation at this site, did not rescue mutant p70s6k activity, indicating that S371 phosphorylation plays an independent role in regulating intrinsic kinase activity.

Amino Acid Sequence↗
PubMed1995

The principal target of rapamycin-induced p70s6k inactivation is a novel phosphorylation site within a conserved hydrophobic domain.

The immunosuppressive agent rapamycin induces inactivation of p70s6k with no effect on other mitogen-activated kinases. Here we have employed a combination of techniques, including mass spectrometry, to demonstrate that this effect is associated with selective dephosphorylation of three previously unidentified p70s6k phosphorylation sites: T229, T389 and S404. T229 resides at a conserved position in the catalytic domain, whose phosphorylation is essential for the activation of other mitogen-induced kinases. However, the principal target of rapamycin-induced p70s6k inactivation is T389, which is located in an unusual hydrophobic sequence outside the catalytic domain. Mutation of T389 to alanine ablates kinase activity, whereas mutation to glutamic acid confers constitutive kinase activity and rapamycin resistance. The importance of this site and its surrounding motif to kinase function is emphasized by its presence in a large number of protein kinases of the second messenger family and its conservation in putative p70s6k homologues from as distantly related organisms as yeast and plants.

3T3 Cells↗
PubMed1995

Rapamycin, wortmannin, and the methylxanthine SQ20006 inactivate p70s6k by inducing dephosphorylation of the same subset of sites.

Activation of p70s6k in cells stimulated with serum correlates with the phosphorylation of seven sites. Pretreatment of Swiss 3T3 cells with the immunosuppressant rapamycin blocks phosphorylation of four of these sites (Thr229, Thr389, Ser404, and Ser411), whereas phosphorylation proceeds in the remaining three sites (Ser418, Thr421, and Ser424). If rapamycin is added postserum stimulation, the pattern of phosphorylation is qualitatively similar except that Ser411 is still highly phosphorylated. The inhibitory effect of rapamycin on serum-induced p70s6k activation and the phosphorylation of Thr229, Thr389, Ser404, and Ser411 is rescued by FK506, providing further evidence that the inhibitory effect is exerted through a complex of rapamycin-FKBP12. Wortmannin treatment pre- or post-serum stimulation inhibits phosphorylation of the same set of sites as rapamycin, supporting the argument that both agents act on the same pathway. Likewise, methylxanthine phosphodiesterase inhibitors block p70s6k activation and phosphorylation of the same set of sites as wortmannin and rapamycin. However, other agents that raise intracellular cAMP levels have no inhibitory effect, leading to the hypothesis that the inhibitory actions of methylxanthines on p70s6k activity are not through activating protein kinase A but through inhibition of an upstream kinase. Together the results indicate that there are two kinase signaling pathways that must converge to activate p70s6k and that only one of these pathways is sensitive to rapamycin, wortmannin, and methylxanthine inhibition.

3T3 Cells↗
PubMed1995

Regulation of p70s6k/p85s6k and its role in the cell cycle.

Two to three-fold increases in the rate of protein synthesis are required both to enter the G1 phase of the cell cycle from G0 and to proceed to S phase in response to growth factors and mitogens. This increase is in part regulated via multiple phosphorylation of the 40S ribosomal protein S6 by the mitogen-stimulated p70s6k/p85s6k. At the protein synthesis level this event appears to be involved in specifically increasing the efficiency of translation of a family of essential mRNAs containing a polypyrimidine tract at their 5' transcriptional start site. The activation of p70s6k/p85s6k and maintenance of its activity throughout G1 is controlled via multiple phosphorylation events mediated by a complex signalling network acting on distinct sets of phosphorylation sites.

Animals↗
PubMed1993

The immunosuppressant rapamycin induces inactivation of p70s6k through dephosphorylation of a novel set of sites.

The immunosuppressant rapamycin selectively abolishes phosphorylation and activation of p70s6k/p85s6k at concentrations that either block or suppress cell growth. The four sites of phosphorylation associated with p70s6k/p85s6k activation all display Ser/Thr-Pro motifs and are closely clustered within a putative autoinhibitory domain of the enzyme. To produce a constitutively active, rapamycin-resistant form of the kinase, these four sites were converted to either Asp or Glu. When overexpressed in human 293 cells, the activity of the mutant is similar to that of the parent enzyme, under conditions where the parent is phosphorylated and active. Unexpectedly, however, the mutant remains sensitive to rapamycin and is inactivated in vitro by protein phosphatase 2A. Peptide maps reveal that rapamycin abolishes the activity of the overexpressed p70s6k through the dephosphorylation of a novel set of sites distinct from those associated with mitogenic activation.

Amino Acid Sequence↗
PubMed1993

Role of the pseudosubstrate sequence in smooth muscle myosin light chain kinase thermal stability.

Smooth muscle myosin light chain kinase (MLCK) is stable in the presence of Ca2+/calmodulin and does not undergo inactivation as reported for skeletal muscle MLCK (Kennelly, P.J., Starovasnik, M.A., Edelman, A.M., and Krebs, E.G. (1990) J. Biol. Chem. 265, 1742-1749). The 61-kDa tryptic fragment of smMLCK-(283-779) with the pseudosubstrate/calmodulin binding sequence deleted undergoes rapid inactivation (t1/2 = 5 min at 25 degrees C). Thermal inactivation renders the 61-kDa fragment more susceptible to cleavage by trypsin. The pseudosubstrate sequence, smMLCK-(787-807) prevents inactivation with high potency (half-maximal protective concentration, PC0.5 = 102 +/- 9 nM). The hexapeptide smMLCK-(797-802), Arg-Arg-Lys-Trp800-Gln-Lys, protected with a similar potency (PC0.5 = 73 +/- 14 nM). The four basic residues as well as Trp were important for maintaining protection by the hexapeptide smMLCK-(797-802). Substitution of Trp800 with Ala or Leu increased the PC0.5 to 500 nM. However, substitution of both aromatic residues Tyr794 and Trp800 in the longer pseudosubstrate peptide-(787-807) had little effect, indicating that with the longer peptide other multiple interactions were sufficient to stabilize the enzyme. The peptide substrate MLC-(11-23) A14,15 was also protective (PC0.5 = 380 nM) as was Mg(2+)-ATP, Mg(2+)-ADP, and Mg2+ plus adenosine. The results demonstrate that the sequence extending from 787-815 encoding the previously identified overlapping pseudosubstrate and calmodulin binding sequences also contains residues that are essential for maintaining thermal stability but these exhibit distinct structure/function relationships.

Amino Acid Sequence↗
PubMed1992

Structural basis of the intrasteric regulation of myosin light chain kinases.

The smooth muscle myosin light chain kinase (smMLCK) catalytic core was modeled by using the crystallographic coordinates of the cyclic AMP-dependent protein kinase catalytic subunit (cAPK) and a bound pseudosubstrate inhibitor peptide, PKI(5-24). Despite only 30% identity in amino acid sequence, the MLCK sequence can be readily accommodated in this structure. With the exception of the short B-helix, all major elements of secondary structure in the core are very likely conserved. The active site of the modeled MLCK complements the known requirements for peptide substrate recognition. MLCK contains a pseudosubstrate sequence that overlaps the calmodulin binding domain and has been proposed to act as an intrasteric inhibitor and occupy the substrate binding site in the absence of Ca(2+)-calmodulin. The pseudosubstrate sequence can be modeled easily into the entire backbone of PKI(5-24). The results demonstrate that the intrasteric model for regulation of MLCK by intramolecular competitive inhibition is structurally plausible.

Amino Acid Sequence↗
PubMed1991

Proteolytic cleavage sites in smooth muscle myosin-light-chain kinase and their relation to structural and regulatory domains.

Proteolysis of the smooth muscle myosin-light-chain kinase with either thermolysin or endoproteinase Lys-C cleaves the enzyme towards the amino-terminus between the first and second unc domains, unc-II-1 and unc-II-2, and in the calmodulin-binding domain. The thermolytic fragment extends 532 residues from Ser275 to Ala806 and is resistant to further digestion. It is catalytically inactive and does not bind calmodulin. Further proteolysis of the thermolytic fragment with trypsin generates a constitutively active fragment. Digestion with endoproteinase Lys-C initially results in an inactive fragment of 516 residues, Ala287 to Lys802. Further digestion with Lys-C endoproteinase results in a constitutively active 474-residue fragment with the same amino-terminus, but a carboxyl-terminus at Lys760, near Arg762, the last conserved residue of protein kinase catalytic domains. There is no cleavage in the acidic-residue-rich connecting peptide between the amino-terminus of the catalytic domain and the unc-I domain, nor within the unc-II or unc-I domains or between the adjacent unc-II-2 and unc-I domains. The pattern of cleavages by these proteases reflects well the predicted domain structure of the myosin-light-chain kinase and further delineates the regulatory pseudosubstrate region. A synthetic peptide corresponding to the pseudosubstrate sequence, MLCK(787-807) was a more potent inhibitor by three orders of magnitude than the overlapping peptide MLCK(777-793) proposed by Ikebe et al. (1989) [Ikebe, M., Maruta, S. & Reardon, S. (1989) J. Biol. Chem. 264, 6967-6971] to be important in autoregulation of the myosin-light-chain kinase.

Amino Acid Sequence↗