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Zahi Damuni - One of the best experts on this subject based on the ideXlab platform.
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Phosphorylation of Eukaryotic Protein Synthesis Initiation Factor 4E by Insulin-stimulated Protamine Kinase
The Journal of biological chemistry, 1995Co-Authors: Anthony Makkinje, Haishan Xiong, Zahi DamuniAbstract:Abstract Insulin-stimulated Protamine Kinase (cPK) and protein Kinase C (PKC) phosphorylated eukaryotic protein synthesis initiation factor 4E (eIF-4E) on serine and threonine residues located on an identical tryptic fragment as judged by two-dimensional phosphopeptide mapping. With cPK and PKC, the apparent K for eIF-4E was about 1.2 and 50 μM, respectively. Relative to recombinant human eIF-4E, cPK exhibited about 100% and ≤5% activity with eIF-4E and eIF-4E, respectively, and eIF-4E was phosphorylated exclusively on threonines. Bovine kidney eIF-4E enhanced up to 1.8-fold globin synthesis in m7GTP-Sepharose-treated reticulocyte lysates. In contrast, following incubation with cPK, these eIF-4E preparations stimulated globin synthesis up to 6-fold. Compared to the dephosphorylation of the cPK-modified serine on eIF-4E, reticulocyte lysates and highly purified protein phosphatase 2A exhibited marked preference for the cPK-modified threonine. The results indicate that cPK phosphorylates eIF-4E on Ser and Thr, that the hydroxyl group or phosphorylation of Thr is necessary for cPK to act on Ser, and that Ser phosphorylation activates reticulocyte globin synthesis. The results suggest that cPK could contribute to the insulin-stimulated phosphorylation of eIF-4E, but that protein phosphatase 2A may confer the site specificity of this response.
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PURIFICATION AND CHARACTERIZATION OF TWO POTENT HEAT-STABLE PROTEIN INHIBITORS OF PROTEIN PHOSPHATASE 2A FROM BOVINE KIDNEY
Biochemistry, 1995Co-Authors: Hong Guo, Zahi DamuniAbstract:Two heat-stable protein inhibitors of protein phosphatase 2A (PP2A), tentatively designated I1PP2A and I2PP2A, have been purified to apparent homogeneity from extracts of bovine kidney. The purified preparations of I1PP2A exhibited an apparent M(r) approximately 30,000 and 250,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel permeation chromatography on Sephacryl S-300, respectively. In contrast, the purified preparations of I2PP2A exhibited an apparent M(r) approximately 20,000 and 80,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel permeation chromatography on Sephacryl S-200, respectively. The purified preparations of I1PP2A and I2PP2A inhibited PP2A with 32P-labeled myelin basic protein, 32P-labeled histone H1, 32P-labeled pyruvate dehydrogenase complex, 32P-labeled phosphorylase, and Protamine Kinase as substrates. By contrast, I1PP2A and I2PP2A exhibited little effect, if any, on the activity of PP2A with 32P-labeled casein, and did not prevent the autodephosphorylation of PP2A in incubations with the autophosphorylation-activated protein Kinase [Guo, H., & Damuni, Z. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 2500-2504]. The purified preparations of I1PP2A and I2PP2A had little effect, if any, on the activities of protein phosphatase 1, protein phosphatase 2B, protein phosphatase 2C, and pyruvate dehydrogenase phosphatase. With 32P-labeled MBP as a substrate, kinetic analysis according to Henderson showed that I1PP2A and I2PP2A were noncompetitive and displayed a Ki of about 30 and 25 nM, respectively. Following cleavage with Staphylococcus aureus V8 protease, I1PP2A and I2PP2A displayed distinct peptide patterns, indicating that these inhibitor proteins are the products of distinct genes. The N-terminal amino acid sequences of the purified preparations indicate that I1PP2A and I2PP2A are novel proteins.
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Autophosphorylation-activated protein Kinase inactivates the protein tyrosine phosphatase activity of protein phosphatase 2A
FEBS letters, 1994Co-Authors: Zahi Damuni, Haishan XiongAbstract:Phosphorylation of the catalytic subunit of protein phosphatase 2A (PP2A) on threonines with a distinct autophosphorylation-activated protein Kinase [Guo and Damuni (1993) Proc. Natl. Acad. Sci. USA 90, 2500-2504] inactivated the phosphatase with 32P-labelled myelin basic protein prepared by incubation with the Kinase domain of the epidermal growth factor receptor, the src-family protein Kinases p56lck and p60c-src, myelin basic protein Kinase-1, or Protamine Kinase. Phosphoamino acid analysis demonstrated that the Kinase domain of the epidermal growth factor receptor, p56lck and p60c-src phosphorylated myelin basic protein on tyrosines, that the Protamine Kinase phosphorylated myelin basic protein on serines, and that myelin basic protein Kinase-1 phosphorylated myelin basic protein on threonines. The results demonstrate that the autophosphorylation-activated protein Kinase not only inactivates the protein serine/threonine phosphatase, but also the protein tyrosine phosphatase activity of PP2A. This autophosphorylation-activated protein Kinase-mediated inactivation of PP2A may, in response to extracellular stimuli, not only contribute to the enhanced phosphorylation of cellular proteins on serines and threonines but also on tyrosines.
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Regulation of in vitro nucleic acid strand annealing activity of heterogeneous nuclear ribonucleoprotein protein A1 by reversible phosphorylation.
Biochemistry, 1994Co-Authors: Haitham Idriss, Zahi Damuni, Hong Guo, Amalendra Kumar, Jose R. Casas-finet, Samuel H. WilsonAbstract:Phosphorylation in vivo of several proteins in the mammalian heterogeneous nuclear ribonucleoprotein complex (hnRNP), including A1, has been observed and proposed as a regulatory step in pre-mRNA splicing [Maryland, S. H., Dwen, P., & Pederson, T. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 7764-7768]. We examined the ability of recombinant hnRNP protein A1 to act as a substrate for a number of purified Ser/Thr protein Kinases in vitro. A survey of seven protein Kinases showed that A1 was heavily phosphorylated by protein Kinase C (PKC) and also was phosphorylated by casein Kinase II, Protamine Kinase, and protein Kinase A. In contrast, autophosphorylation-activated protein Kinase and two forms of myelin basic protein Kinase failed to phosphorylate A1. Proteolysis with trypsin and V8 protease revealed that PKC phosphorylates A1 at three main sites, two in the N-terminal domain (spanning residues 2-196) and one in the C-terminal domain (spanning residues 197-320). Amino acid sequencing revealed that these sites were Ser95, Ser192, and Ser199; phosphorylation at Ser192 was more abundant than at Ser95 and Ser199. Phosphorylation by PKC inhibited the strand annealing activity of A1. Protein phosphatase 2A, but not protein phosphatase 1, dephosphorylated A1 and reversed the inhibitory effect of PKC phosphorylation on the strand annealing activity. A conformational change in the C-terminal domain of A1 was observed upon PKC phosphorylation, and this was associated with a decrease in A1's affinity for single-stranded polynucleotides. The results are consistent with a role of phosphorylation of A1 in regulating its strand annealing activity in vivo.
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Phosphorylation and activation of Protamine Kinase by two forms of a myelin basic protein Kinase from extracts of bovine kidney cortex.
The Journal of biological chemistry, 1993Co-Authors: S. A. G. Reddy, Hong Guo, S. Z. Tarun, Zahi DamuniAbstract:Two myelin basic protein Kinases designated MBPK-1 and MBPK-2 were purified to apparent homogeneity from extracts of bovine kidney cortex. The purified preparations exhibited an apparent M(r) approximately 40,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and approximately 42,000 (MBPK-1) and 45,000 (MBPK-2) by gel permeation chromatography. Up to 0.4 and 1.8 mol of phosphoryl groups were incorporated per mol of MBPK-1 and MBPK-2, respectively, on threonines following incubation with ATP. Autophosphorylation, incubation with protein phosphatase 2A2 (PP2A2), CD45, or T-cell protein tyrosine phosphatase did not affect MBPK-1 activity. Autophosphorylation increased by about 3-fold MBPK-2 activity. This autophosphorylation and activation was reversed by PP2A2 but not by CD45 or T-cell protein tyrosine phosphatase. MBPK-1 and MBPK-2 displayed a positive reaction with an antibody to mitogen-activated protein Kinase. Purified preparations of Protamine Kinase were activated by about 1.5-6-fold and, after inactivation with PP2A2, were reactivated by about 30% by MBPK-1 and MBPK-2. Activation and reactivation correlated with the incorporation, respectively, of 0.1-0.5 and 0.5 mol of phosphoryl groups/mol of the Protamine Kinase on serines. The results show that MBPK-1 and MBPK-2 are Protamine Kinase-activating Kinases and suggest that MBPK-1 and MBPK-2 may be related to mitogen-activated protein Kinase.
Grayson D. Amick - One of the best experts on this subject based on the ideXlab platform.
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Purification and properties of a Protamine Kinase from bovine kidney microsomes.
Archives of biochemistry and biophysics, 1992Co-Authors: Grayson D. Amick, S. A. G. Reddy, Zahi DamuniAbstract:Abstract About an eightfold increase in Protamine Kinase activity was detected following extraction of highly purified microsomes from bovine kidney with 1% Triton X-100. Relative to the soluble fraction, the microsomes contained about 30% Protamine Kinase activity. The microsomal Protamine Kinase was purified to apparent homogeneity. The purified enzyme exhibited an apparent M r ≈ 45,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel permeation chromatography on Sephacryl S-200. Relative to Protamine, the purified Kinase exhibited about 100% activity with the synthetic peptide RRLSSLRA and about 5,8, and 2 , purified preparations of the Protamine Kinase were inactivated. These properties were identical to those of purified preparations of a Protamine Kinase from extracts of bovine kidney cytosol ( Z. Damuni, G. D. Amick, and T. R. Sneed, 1989 , J. Biol. Chem. 264, 6412–6418). Near identical peptide patterns were obtained following incubation of purified preparations of the microsomal and cytosolic Protamine Kinases with Staphylococcus aureus V8 proteinase. The results indicate that a form of the cytosolic Protamine Kinase is present in microsomes.
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Protamine Kinase phosphorylates eukaryotic protein synthesis initiation factor 4E.
Biochemical and biophysical research communications, 1992Co-Authors: Grayson D. Amick, Zahi DamuniAbstract:Up to 1 mol of phosphoryl groups was incorporated per mol of eukaryotic protein synthesis initiation factor (eIF) 4E following incubation of purified preparations of this factor with purified preparations of a Protamine Kinase from bovine kidney cytosol. By contrast, purified preparations of two forms of mitogen-activated protein Kinase, casein Kinase II and two forms of a distinct autophosphorylation-activated protein Kinase exhibited little activity, if any, with eIF-4E. Together with previous observations, the results indicate that the Protamine Kinase could contribute to the insulin-stimulated phosphorylation of eIF-4E.
Hong Guo - One of the best experts on this subject based on the ideXlab platform.
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PURIFICATION AND CHARACTERIZATION OF TWO POTENT HEAT-STABLE PROTEIN INHIBITORS OF PROTEIN PHOSPHATASE 2A FROM BOVINE KIDNEY
Biochemistry, 1995Co-Authors: Hong Guo, Zahi DamuniAbstract:Two heat-stable protein inhibitors of protein phosphatase 2A (PP2A), tentatively designated I1PP2A and I2PP2A, have been purified to apparent homogeneity from extracts of bovine kidney. The purified preparations of I1PP2A exhibited an apparent M(r) approximately 30,000 and 250,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel permeation chromatography on Sephacryl S-300, respectively. In contrast, the purified preparations of I2PP2A exhibited an apparent M(r) approximately 20,000 and 80,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel permeation chromatography on Sephacryl S-200, respectively. The purified preparations of I1PP2A and I2PP2A inhibited PP2A with 32P-labeled myelin basic protein, 32P-labeled histone H1, 32P-labeled pyruvate dehydrogenase complex, 32P-labeled phosphorylase, and Protamine Kinase as substrates. By contrast, I1PP2A and I2PP2A exhibited little effect, if any, on the activity of PP2A with 32P-labeled casein, and did not prevent the autodephosphorylation of PP2A in incubations with the autophosphorylation-activated protein Kinase [Guo, H., & Damuni, Z. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 2500-2504]. The purified preparations of I1PP2A and I2PP2A had little effect, if any, on the activities of protein phosphatase 1, protein phosphatase 2B, protein phosphatase 2C, and pyruvate dehydrogenase phosphatase. With 32P-labeled MBP as a substrate, kinetic analysis according to Henderson showed that I1PP2A and I2PP2A were noncompetitive and displayed a Ki of about 30 and 25 nM, respectively. Following cleavage with Staphylococcus aureus V8 protease, I1PP2A and I2PP2A displayed distinct peptide patterns, indicating that these inhibitor proteins are the products of distinct genes. The N-terminal amino acid sequences of the purified preparations indicate that I1PP2A and I2PP2A are novel proteins.
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Regulation of in vitro nucleic acid strand annealing activity of heterogeneous nuclear ribonucleoprotein protein A1 by reversible phosphorylation.
Biochemistry, 1994Co-Authors: Haitham Idriss, Zahi Damuni, Hong Guo, Amalendra Kumar, Jose R. Casas-finet, Samuel H. WilsonAbstract:Phosphorylation in vivo of several proteins in the mammalian heterogeneous nuclear ribonucleoprotein complex (hnRNP), including A1, has been observed and proposed as a regulatory step in pre-mRNA splicing [Maryland, S. H., Dwen, P., & Pederson, T. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 7764-7768]. We examined the ability of recombinant hnRNP protein A1 to act as a substrate for a number of purified Ser/Thr protein Kinases in vitro. A survey of seven protein Kinases showed that A1 was heavily phosphorylated by protein Kinase C (PKC) and also was phosphorylated by casein Kinase II, Protamine Kinase, and protein Kinase A. In contrast, autophosphorylation-activated protein Kinase and two forms of myelin basic protein Kinase failed to phosphorylate A1. Proteolysis with trypsin and V8 protease revealed that PKC phosphorylates A1 at three main sites, two in the N-terminal domain (spanning residues 2-196) and one in the C-terminal domain (spanning residues 197-320). Amino acid sequencing revealed that these sites were Ser95, Ser192, and Ser199; phosphorylation at Ser192 was more abundant than at Ser95 and Ser199. Phosphorylation by PKC inhibited the strand annealing activity of A1. Protein phosphatase 2A, but not protein phosphatase 1, dephosphorylated A1 and reversed the inhibitory effect of PKC phosphorylation on the strand annealing activity. A conformational change in the C-terminal domain of A1 was observed upon PKC phosphorylation, and this was associated with a decrease in A1's affinity for single-stranded polynucleotides. The results are consistent with a role of phosphorylation of A1 in regulating its strand annealing activity in vivo.
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Phosphorylation and activation of Protamine Kinase by two forms of a myelin basic protein Kinase from extracts of bovine kidney cortex.
The Journal of biological chemistry, 1993Co-Authors: S. A. G. Reddy, Hong Guo, S. Z. Tarun, Zahi DamuniAbstract:Two myelin basic protein Kinases designated MBPK-1 and MBPK-2 were purified to apparent homogeneity from extracts of bovine kidney cortex. The purified preparations exhibited an apparent M(r) approximately 40,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and approximately 42,000 (MBPK-1) and 45,000 (MBPK-2) by gel permeation chromatography. Up to 0.4 and 1.8 mol of phosphoryl groups were incorporated per mol of MBPK-1 and MBPK-2, respectively, on threonines following incubation with ATP. Autophosphorylation, incubation with protein phosphatase 2A2 (PP2A2), CD45, or T-cell protein tyrosine phosphatase did not affect MBPK-1 activity. Autophosphorylation increased by about 3-fold MBPK-2 activity. This autophosphorylation and activation was reversed by PP2A2 but not by CD45 or T-cell protein tyrosine phosphatase. MBPK-1 and MBPK-2 displayed a positive reaction with an antibody to mitogen-activated protein Kinase. Purified preparations of Protamine Kinase were activated by about 1.5-6-fold and, after inactivation with PP2A2, were reactivated by about 30% by MBPK-1 and MBPK-2. Activation and reactivation correlated with the incorporation, respectively, of 0.1-0.5 and 0.5 mol of phosphoryl groups/mol of the Protamine Kinase on serines. The results show that MBPK-1 and MBPK-2 are Protamine Kinase-activating Kinases and suggest that MBPK-1 and MBPK-2 may be related to mitogen-activated protein Kinase.
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Autophosphorylation-activated protein Kinase phosphorylates and inactivates protein phosphatase 2A
Proceedings of the National Academy of Sciences of the United States of America, 1993Co-Authors: Hong Guo, Zahi DamuniAbstract:Purified preparations of a distinct autophosphorylation-activated protein Kinase from bovine kidney phosphorylated and inactivated purified preparations of protein phosphatase 2A2 (PP2A2) by about 80% with the autophosphorylation-activated protein Kinase, Protamine Kinase, and 32P-labeled myelin basic protein as substrates. Analysis of incubations performed in the presence of 0.2 mM [gamma-32P]ATP by autoradiography following SDS/PAGE and by FPLC gel permeation chromatography on Superose 12 demonstrated that the catalytic subunit of PP2A2 was phosphorylated in the incubation mixtures containing the Kinase and phosphatase. Up to 0.3 mol of phosphate groups was incorporated per mol of the catalytic subunit of PP2A2 following incubation with the Kinase. This phosphorylation was enhanced about 5-fold in the presence of 0.4 microM microcystin-LR. In addition, up to 1 mol of phosphate groups was incorporated per mol of the PP2A2 subunit of apparent M(r) approximately 60,000 when microcystin-LR was included. Analysis by thin-layer chromatography indicated that PP2A2 catalyzed an autodephosphorylation reaction which was inhibited by microcystin-LR. Phospho amino acid analysis showed that the catalytic subunit of PP2A2 was phosphorylated on threonine residues by the autophosphorylation-activated protein Kinase. Together with previous observations, the results suggest that inactivation of PP2A by phosphorylation catalyzed by the autophosphorylation-activated protein Kinase could contribute to the marked increase in the phosphorylation of cellular proteins in response to insulin and other mitogens.
Haishan Xiong - One of the best experts on this subject based on the ideXlab platform.
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Phosphorylation of Eukaryotic Protein Synthesis Initiation Factor 4E by Insulin-stimulated Protamine Kinase
The Journal of biological chemistry, 1995Co-Authors: Anthony Makkinje, Haishan Xiong, Zahi DamuniAbstract:Abstract Insulin-stimulated Protamine Kinase (cPK) and protein Kinase C (PKC) phosphorylated eukaryotic protein synthesis initiation factor 4E (eIF-4E) on serine and threonine residues located on an identical tryptic fragment as judged by two-dimensional phosphopeptide mapping. With cPK and PKC, the apparent K for eIF-4E was about 1.2 and 50 μM, respectively. Relative to recombinant human eIF-4E, cPK exhibited about 100% and ≤5% activity with eIF-4E and eIF-4E, respectively, and eIF-4E was phosphorylated exclusively on threonines. Bovine kidney eIF-4E enhanced up to 1.8-fold globin synthesis in m7GTP-Sepharose-treated reticulocyte lysates. In contrast, following incubation with cPK, these eIF-4E preparations stimulated globin synthesis up to 6-fold. Compared to the dephosphorylation of the cPK-modified serine on eIF-4E, reticulocyte lysates and highly purified protein phosphatase 2A exhibited marked preference for the cPK-modified threonine. The results indicate that cPK phosphorylates eIF-4E on Ser and Thr, that the hydroxyl group or phosphorylation of Thr is necessary for cPK to act on Ser, and that Ser phosphorylation activates reticulocyte globin synthesis. The results suggest that cPK could contribute to the insulin-stimulated phosphorylation of eIF-4E, but that protein phosphatase 2A may confer the site specificity of this response.
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Autophosphorylation-activated protein Kinase inactivates the protein tyrosine phosphatase activity of protein phosphatase 2A
FEBS letters, 1994Co-Authors: Zahi Damuni, Haishan XiongAbstract:Phosphorylation of the catalytic subunit of protein phosphatase 2A (PP2A) on threonines with a distinct autophosphorylation-activated protein Kinase [Guo and Damuni (1993) Proc. Natl. Acad. Sci. USA 90, 2500-2504] inactivated the phosphatase with 32P-labelled myelin basic protein prepared by incubation with the Kinase domain of the epidermal growth factor receptor, the src-family protein Kinases p56lck and p60c-src, myelin basic protein Kinase-1, or Protamine Kinase. Phosphoamino acid analysis demonstrated that the Kinase domain of the epidermal growth factor receptor, p56lck and p60c-src phosphorylated myelin basic protein on tyrosines, that the Protamine Kinase phosphorylated myelin basic protein on serines, and that myelin basic protein Kinase-1 phosphorylated myelin basic protein on threonines. The results demonstrate that the autophosphorylation-activated protein Kinase not only inactivates the protein serine/threonine phosphatase, but also the protein tyrosine phosphatase activity of PP2A. This autophosphorylation-activated protein Kinase-mediated inactivation of PP2A may, in response to extracellular stimuli, not only contribute to the enhanced phosphorylation of cellular proteins on serines and threonines but also on tyrosines.
Anthony Makkinje - One of the best experts on this subject based on the ideXlab platform.
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Phosphorylation of Eukaryotic Protein Synthesis Initiation Factor 4E by Insulin-stimulated Protamine Kinase
The Journal of biological chemistry, 1995Co-Authors: Anthony Makkinje, Haishan Xiong, Zahi DamuniAbstract:Abstract Insulin-stimulated Protamine Kinase (cPK) and protein Kinase C (PKC) phosphorylated eukaryotic protein synthesis initiation factor 4E (eIF-4E) on serine and threonine residues located on an identical tryptic fragment as judged by two-dimensional phosphopeptide mapping. With cPK and PKC, the apparent K for eIF-4E was about 1.2 and 50 μM, respectively. Relative to recombinant human eIF-4E, cPK exhibited about 100% and ≤5% activity with eIF-4E and eIF-4E, respectively, and eIF-4E was phosphorylated exclusively on threonines. Bovine kidney eIF-4E enhanced up to 1.8-fold globin synthesis in m7GTP-Sepharose-treated reticulocyte lysates. In contrast, following incubation with cPK, these eIF-4E preparations stimulated globin synthesis up to 6-fold. Compared to the dephosphorylation of the cPK-modified serine on eIF-4E, reticulocyte lysates and highly purified protein phosphatase 2A exhibited marked preference for the cPK-modified threonine. The results indicate that cPK phosphorylates eIF-4E on Ser and Thr, that the hydroxyl group or phosphorylation of Thr is necessary for cPK to act on Ser, and that Ser phosphorylation activates reticulocyte globin synthesis. The results suggest that cPK could contribute to the insulin-stimulated phosphorylation of eIF-4E, but that protein phosphatase 2A may confer the site specificity of this response.
