A membrane-bound protein kinase from rabbit reticulocytes is an active form of multipotential S6 kinase

Bensen, Eric S.; Umphress, Jason L.; Traugh, Jolinda A.; Pinna, Lorenzo A.; Tuazon, Polygena T.

doi:10.1016/0167-4838(95)00209-x

Cited by 4 publications

(6 citation statements)

References 35 publications

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“…Peptides containing the sequence X(K/R)RXSAA, with X as alanine or a basic amino acid, varied only slightly in the measured K m and V max values. The K m values (0.37-0.65 mM) were within the range observed with other protein kinases for synthetic peptides (30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45); the V max values varied from 169 to 750 pmol min -1 µg -1 . Peptides with a third basic amino acid in the -1 or -4 position had the highest overall rate as shown by the V max /K m value, ∼2-fold higher than that of AKRASAA.…”

Section: Resultssupporting

confidence: 78%

“…Assays with peptides (1 mM) were carried out with γ-PAK (1.6-5.6 units) in 25 µL reaction mixtures incubated for 30 min at 30 °C under conditions described above, except that 0.4 mg/mL of bovine serum albumin was added and the specific activity of ATP was 300-2000 cpm/pmol. Reactions were terminated with 5 µL of 100 mM ATP, and the amount of 32 P incorporated was quantitated by precipitation of 20 µL aliquots on P81 phosphocellulose paper with 75 mM H 3 PO 4 as described previously (30). Peptides containing only one basic amino acid were analyzed by thin-layer electrophoresis on cellulose sheets for 2 h at 600 V with pyridine/acetic acid/water (10:100:890), pH 3.5, as solvent, as described previously (30).…”

Section: Methodsmentioning

confidence: 99%

“…Reactions were terminated with 5 µL of 100 mM ATP, and the amount of 32 P incorporated was quantitated by precipitation of 20 µL aliquots on P81 phosphocellulose paper with 75 mM H 3 PO 4 as described previously (30). Peptides containing only one basic amino acid were analyzed by thin-layer electrophoresis on cellulose sheets for 2 h at 600 V with pyridine/acetic acid/water (10:100:890), pH 3.5, as solvent, as described previously (30). Activation of γ-PAK by autophosphorylation in the presence of Cdc42(GTPγS) was performed as previously described (7).…”

Section: Methodsmentioning

confidence: 99%

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Determinants for Substrate Phosphorylation by p21-Activated Protein Kinase (γ-PAK)

et al. 1997

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gamma-PAK, originally designated PAK I and subsequently identified as a member of the p21-activated protein kinase family, has been shown to have cytostatic properties and to be involved in maintaining cells in a nondividing state [Rooney, R. D., et al., (1996) J. Biol. Chem. 271, 21498-21504]. The determinants for phosphorylation of substrates by gamma-PAK have been identified by examining the kinetics of phosphorylation of a series of synthetic peptides patterned after the sequence KKRKSGL, which is the site phosphorylated by gamma-PAK in the Rous sarcoma virus nucleocapsid protein NC in vivo and in vitro. With these peptides, the recognition sequence for gamma-PAK has been shown to contain two basic amino acids in the -2 and -3 positions, as represented by (K/R)RXS, in which the -2 position is an arginine, the -3 position is an arginine or a lysine, and X can be an acidic, basic, or neutral amino acid. A basic amino acid in the -1 or -4 position improves the rate of phosphorylation by increasing the Vmax and decreasing the Km. An acidic amino acid in the -1 position increases the rate (2.5-fold), as does an acidic residue in the -4 position, although to a lower extent (1.6-fold). Proline in the -1 or +1 position has a deleterious effect and inhibits phosphorylation by gamma-PAK. The substrate requirements of protein kinases that recognize basic amino acids on the N-terminal side of the phosphorylatable residue such as cAMP-dependent protein kinase (PKA) and Ca2+/phospholipid-dependent protein kinase (PKC) have been compared with gamma-PAK using the same peptides. An acidic residue in the -1 position negatively affects PKA and PKC; thus, peptides containing the sequence KRES can be used to identify gamma-PAK.

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Section: Resultssupporting

confidence: 78%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Determinants for Substrate Phosphorylation by p21-Activated Protein Kinase (γ-PAK)

et al. 1997

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“…Phosphorylation of peptide S3 (18 µg) was carried out under the same conditions with 0.1 µg of γ‐PAK, 0.5 µg of Cdc42, and 25 µL reaction mixtures; incubation was for 30 min. Reactions were terminated by addition of nonlabeled ATP and analyzed by precipitation on P81 phosphocellulose paper as described previously [34].…”

Section: Methodsmentioning

confidence: 99%

Substrates enhance autophosphorylation and activation of p21‐activated protein kinase γ‐PAK in the absence of activation loop phosphorylation

Jakobi

Huang

Walter

et al. 2000

European Journal of Biochemistry

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The p21‐activated protein kinase γ‐PAK from rabbit, expressed in insect cells, is activated following binding of Cdc42(GTPγS). The rate of autophosphorylation is increased fivefold and the protein kinase activity 13‐fold, as measured with the synthetic heptapeptide (AKRESAA). The mutant K278R, where the invariant lysine in the catalytic site is replaced by arginine, shows neither autophosphorylation nor activity. Replacement of the conserved threonine in the catalytic domain with alanine (T402A) reduces autophosphorylation and protein kinase activity to 1% that of the wild‐type γ‐PAK, indicating autophosphorylation of Thr402 in the activation loop is essential for protein kinase activity. In contrast, certain protein substrates such as histone 2B, histone 4 and myelin basic protein, stimulate both autophosphorylation and protein kinase activity to levels similar to those observed with Cdc42(GTPγS). This substrate‐level activation does not require autophosphorylation of Thr402 in the activation loop. As shown with T402A, the protein kinase activity with histone 4 is similar to that observed with recombinant wild‐type γ‐PAK. Basic proteins or peptides which are not substrates of γ‐PAK, such as histone 1 and polylysine, do not stimulate autophosphorylation or activity. Other substrates such as the Rous sarcoma virus protein NC are phosphorylated by γ‐PAK following activation by Cdc42(GTPγS), but are not phosphorylated by T402A. The data suggest that some substrates can override the requirement for Cdc42(GTPγS), by activating γ‐PAK directly.

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“…Multipotential S6 kinase was first purified from the cytosol of rabbit reticulocytes as an inactive enzyme that could be activated by limited proteolysis in vitro (21,22). An endogenously active form has been purified from the cytosol of rabbit liver (23) as well as from the membrane of rabbit reticulocytes (24). Multipotential S6 kinase activity is stimulated in 3T3-L1 cells in response to insulin (20,25) and is different from the type I and II S6 kinases (p90 rsk and p70 s6k ) (26), as shown by phosphorylation of a number of other substrates including histone 1 (21,22), initiation factors (eIF-2, eIF-3, eIF-4B and eIF-4F) (27), and glycogen synthase (28).…”

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confidence: 99%

Phosphorylation of Elongation Factor 1 and Ribosomal Protein S6 by Multipotential S6 Kinase and Insulin Stimulation of Translational Elongation

Chang

Traugh

1997

Journal of Biological Chemistry

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Stimulation of protein synthesis in response to insulin is concomitant with increased phosphorylation of initiation factors 4B and 4G and ribosomal protein S6 (Morley, S. J., and Traugh, J. A. (1993) Biochimie 75, 985-989) and is due at least in part to multipotential S6 kinase. When elongation factor 1 (EF-1) from rabbit reticulocytes was examined as substrate for multipotential S6 kinase, up to 1 mol/mol of phosphate was incorporated into the ␣, ␤, and ␦ subunits. Phosphorylation of EF-1 resulted in a 2-2.6-fold stimulation of EF-1 activity, as measured by poly(U)-directed polyphenylalanine synthesis. The rate of elongation was also stimulated by approximately 2-fold with 80 S ribosomes phosphorylated on S6 by multipotential S6 kinase. When the rates of elongation in extracts from serum-fed 3T3-L1 cells and cells serum-deprived for 1.5 h were compared, a 40% decrease was observed upon serum deprivation. The addition of insulin to serum-deprived cells for 15 min stimulated elongation to a rate equivalent to that of serumfed cells. Similar results were obtained with partially purified EF-1, with both EF-1 and ribosomes contributing to stimulation of elongation. These data are consistent with a ribosomal transit time of 3.2 min for serumdeprived cells and 1.6 min following the addition of insulin for 15 min. Taken together, the data suggest that insulin stimulation involves coordinate regulation of EF-1 and ribosomes through phosphorylation by multipotential S6 kinase. Elongation factor 1 (EF-1)1 is composed of a nucleotide-binding protein, EF-1␣, and a nucleotide exchange protein complex, EF-1␤␥␦ (1, 2). The EF-1␣ subunit (50.1 kDa) binds GTP and mediates the attachment of aminoacyl-tRNA to ribosomes (1, 2), while the ␤ (24.8-kDa), ␥ (50.0-kDa), and ␦ (31.1-kDa) subunits stimulate the exchange of GDP for GTP on the ␣ subunit (3-5). Several forms of EF-1 can be isolated from eukaryotic cells, including ␣, ␣␤␥, and ␣␤␥␦ (6 -8). Approximately 15% of the EF-1␣␤␥␦ is complexed with valyl-tRNA synthetase (ValRS) (7-11).The mechanisms involved in the regulation of EF-1 activity are not well understood, although EF-1 activity has been shown to be modulated by mitogens, aging, heat shock, and fertilization (12). All four subunits of EF-1 have been shown to undergo phosphorylation in vivo and in vitro. When the ␣, ␤, and ␦ subunits are phosphorylated in rabbit reticulocytes in response to phorbol 12-myristate 13-acetate, a 2-3-fold stimulation of EF-1 activity is observed (6). A similar effect is observed when EF-1 is phosphorylated in vitro by protein kinase C (7), and this effect is due to stimulation of the rate-limiting step, GTP/GDP exchange on EF-1␣ (8).The ␤ and ␦ subunits are also phosphorylated by protein kinase CKII (13-16). GDP has been shown to enhance the phosphorylation of ␤ and ␦ by CKII, while little phosphorylation is observed in the absence of GDP (16). In addition, the ␥ and ␦ subunits of EF-1 from Xenopus oocytes are substrates for the cell cycle division control kinase (Cdc2) (17, 18). The physiologic...

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A membrane-bound protein kinase from rabbit reticulocytes is an active form of multipotential S6 kinase

Cited by 4 publications

References 35 publications

Determinants for Substrate Phosphorylation by p21-Activated Protein Kinase (γ-PAK)

Determinants for Substrate Phosphorylation by p21-Activated Protein Kinase (γ-PAK)

Substrates enhance autophosphorylation and activation of p21‐activated protein kinase γ‐PAK in the absence of activation loop phosphorylation

Phosphorylation of Elongation Factor 1 and Ribosomal Protein S6 by Multipotential S6 Kinase and Insulin Stimulation of Translational Elongation

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