Cyclic AMP-resistant mutants of S49 mouse lymphoma cells hemizygous for expression of regulatory subunit of type I cyclic AMP-dependent protein kinase

1987 ◽  
Vol 13 (6) ◽  
pp. 645-659 ◽  
Author(s):  
Robert A. Steinberg ◽  
Caroline S. Murphy ◽  
Joanne L. Russell ◽  
Karen B. German
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Regulatory Subunit ◽  
Type I ◽  
Lymphoma Cells ◽  
Dependent Protein Kinase ◽  
Mouse Lymphoma Cells ◽  
Dependent Protein ◽  
Resistant Mutants ◽  
Mouse Lymphoma

scholarly journals Sites of phosphorylation and mutation in regulatory subunit of cyclic AMP-dependent protein kinase from S49 mouse lymphoma cells: mapping to structural domains.

1983 ◽  
Vol 97 (4) ◽  
pp. 1072-1080 ◽  
Author(s):  
R A Steinberg
Keyword(s):  
Protein Kinase ◽  
Hinge Region ◽  
Regulatory Subunit ◽  
Type I ◽  
Lymphoma Cells ◽  
Dependent Protein Kinase ◽  
Structural Domains ◽  
Mouse Lymphoma Cells ◽  
Dependent Protein ◽  
Mouse Lymphoma

A novel peptide mapping approach has been used to map sites of charge modification to major structural domains of regulatory subunit (R) of type I cAMP-dependent protein kinase from S49 mouse lymphoma cells. Proteolytic fragments of crude, radiolabeled R were purified by cAMP affinity chromatography and displayed by two-dimensional polyacrylamide gel electrophoresis. [35S]methionine-labeled peptides containing sites of mutation or phosphorylation exhibited charge heterogeneity attributable to the modification. Phosphate-containing fragments were also labeled with [32P]orthophosphate to confirm their phosphorylation. Major fragments from [35S]methionine-labeled S49 cell R corresponded in size to carboxyterminal cAMP-binding fragments reported from proteolysis of purified type I Rs from various mammalian species; additional fragments were also visualized. End-specific markers in Rs from some mutant S49 sublines confirmed that cAMP-binding fragments extended to the carboxyterminus of R. Aminoterminal endpoints of fragments could be deduced, therefore, from peptide molecular weights. Clustering of proteolytic cleavage sites within the "hinge-region" separating aminoterminal and carboxyterminal domains of R permitted high resolution mapping in this region: the endogenous phosphate and a "phenotypically-silent" electrophoretic marker mutation fell within a 2.5-kdalton interval at its aminoterminal end. On the other hand, Ka mutations that increase the apparent constant for activation of kinase by cAMP mapped within the large cAMP-binding region of R. A map of charge density distribution within the hinge-region of R was constructed to facilitate structural comparisons between Rs from S49 cells and from other mammalian sources.

scholarly journals A kinase-negative mutant of S49 mouse lymphoma cells is defective in posttranslational maturation of catalytic subunit of cyclic AMP-dependent protein kinase.

1991 ◽  
Vol 11 (2) ◽  
pp. 705-712 ◽  
Author(s):  
R A Steinberg
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Sodium Dodecyl ◽  
Wild Type ◽  
Lymphoma Cells ◽  
Dependent Protein Kinase ◽  
Mouse Lymphoma Cells ◽  
C Subunit ◽  
Dependent Protein ◽  
Mouse Lymphoma

Kinase-negative mutants of S49 mouse lymphoma cells, which lack detectable catalytic (C) subunit of cyclic AMP-dependent protein kinase, nevertheless contain cytoplasmic mRNAs for the two major forms of C subunit, C alpha and C beta. Investigation of the metabolism of C subunits in wild-type and mutant cells was undertaken to identify the step(s) at which C subunit expression was defective in kinase-negative cells. [35S]methionine-labeled C subunits from cytosolic fractions of wild-type S49 cells or C subunit-overexpressing cell lines were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after purification by either affinity chromatography using a peptide inhibitor of C subunit as the ligand or immunoadsorption with an anti-C subunit antiserum. Immunoadsorption revealed electrophoretic forms of C alpha and C beta subunits that migrated faster than those detected in affinity-purified samples; this unexpected heterogeneity suggested that functional activation of C subunit may require posttranslational modification. Immunoadsorption of cytosolic fractions from wild-type cells labeled for various times with [35S]methionine revealed an additional posttranslational maturation step. The bulk of immunoadsorbable C subunit label in cells pulse-labeled for 5 min or less was in an insoluble fraction from which it could be solubilized with a detergent-containing buffer; solubilization of the newly synthesized material proceeded over an incubation period of about 10 min. The primary defect in kinase-negative cells appeared to be in this solubilization step, since about equal C subunit radioactivity was found in detergent extracts of wild-type and kinase-negative cells but very little was found in mutant cytosols. I speculate that an accessory factor required for proper folding of newly synthesized C subunit in defective in the kinase-negative cells.

A kinase-negative mutant of S49 mouse lymphoma cells is defective in posttranslational maturation of catalytic subunit of cyclic AMP-dependent protein kinase

1991 ◽  
Vol 11 (2) ◽  
pp. 705-712
Author(s):  
R A Steinberg
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Sodium Dodecyl ◽  
Wild Type ◽  
Lymphoma Cells ◽  
Dependent Protein Kinase ◽  
Mouse Lymphoma Cells ◽  
C Subunit ◽  
Dependent Protein ◽  
Mouse Lymphoma

Kinase-negative mutants of S49 mouse lymphoma cells, which lack detectable catalytic (C) subunit of cyclic AMP-dependent protein kinase, nevertheless contain cytoplasmic mRNAs for the two major forms of C subunit, C alpha and C beta. Investigation of the metabolism of C subunits in wild-type and mutant cells was undertaken to identify the step(s) at which C subunit expression was defective in kinase-negative cells. [35S]methionine-labeled C subunits from cytosolic fractions of wild-type S49 cells or C subunit-overexpressing cell lines were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after purification by either affinity chromatography using a peptide inhibitor of C subunit as the ligand or immunoadsorption with an anti-C subunit antiserum. Immunoadsorption revealed electrophoretic forms of C alpha and C beta subunits that migrated faster than those detected in affinity-purified samples; this unexpected heterogeneity suggested that functional activation of C subunit may require posttranslational modification. Immunoadsorption of cytosolic fractions from wild-type cells labeled for various times with [35S]methionine revealed an additional posttranslational maturation step. The bulk of immunoadsorbable C subunit label in cells pulse-labeled for 5 min or less was in an insoluble fraction from which it could be solubilized with a detergent-containing buffer; solubilization of the newly synthesized material proceeded over an incubation period of about 10 min. The primary defect in kinase-negative cells appeared to be in this solubilization step, since about equal C subunit radioactivity was found in detergent extracts of wild-type and kinase-negative cells but very little was found in mutant cytosols. I speculate that an accessory factor required for proper folding of newly synthesized C subunit in defective in the kinase-negative cells.

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