Expression in Escherichia coli and characterization of the heat-stable inhibitor of the cAMP-dependent protein kinase

Pure heat-stable inhibitor of the cAMP-dependent protein kinase (PKI) has been isolated in high yield by using a bacterial expression vector constructed to synthesize the complete sequence of the rabbit muscle protein kinase inhibitor, plus an amino-terminal initiator methionine and glycine. Bacterially expressed PKI has an inhibitory activity identical to that of the protein isolated from rabbit skeletal muscle and, by gel filtration and gel electrophoresis, has the same physicochemical characteristics as the native physiological form of PKI. Fourier transformed infrared spectroscopy and CD establish that PKI has unusually large amounts of random coil and turn structures, with significantly smaller amounts of alpha-helix and beta structures.     

Reproductive function in protein kinase inhibitor-deficient mice

The protein kinase inhibitor (PKI) family includes three genes encoding small, heat-stable inhibitors of the cyclic AMP-dependent kinase PKA. Each PKI isoform contains a PKA inhibitory domain and a nuclear export domain, enabling PKI to both inhibit PKA and remove it from the nucleus. The PKIbeta isoform, also known as testis PKI, is highly expressed in germ cells of the testis and is found at more modest levels in other tissues. In order to investigate its physiological role, we have generated PKIbeta knockout mice by gene targeting. These mice exhibit a partial loss of PKI activity in testis but remain fertile with normal testis development and function. PKIbeta knockout females also reproduce normally. The PKIbeta mutants were crossed with our previously derived PKIalpha mutants to obtain double-knockout mice. Remarkably, these mice are also viable and fertile with no obvious physiological defects in either males or females.     

A novel conformationally restricted protein kinase C inhibitor, Ro 31-8425, inhibits human neutrophil superoxide generation by soluble, particulate and post-receptor stimuli.

A novel, bis-indolylmaleimide, Ro 31-8425, bearing a conformationally restricted side chain, inhibits protein kinase C isolated from rat brain and human neutrophils with a high degree of selectivity over cAMP-dependent kinase and Ca2+/calmodulin-dependent kinase. It also inhibits phorbol ester-induced intracellular events known to be mediated by protein kinase C (p47 phosphorylation in intact platelets, CD3 and CD4 down-regulation in T-cells). Ro 31-8425 inhibited superoxide generation in human neutrophils activated by both receptor stimuli (formyl-methionyl-leucylphenylalanine, opsonized zymosan, IgG and heat aggregated IgG) and post-receptor stimuli (1,2-dioctanoylglycerol and fluoride). The compound also blocked antigen driven, but not IL-2 induced, T-cell proliferation. These results support a central role for protein kinase C in the activation of the respiratory burst and antigen-driven T-cell proliferation.     

Concerted phosphorylation of the 26-kilodalton phospholamban oligomer and of the low molecular weight phospholamban subunits

Phospholamban (PLB) from cardiac sarcoplasmic reticulum (SR) was phosphorylated under various conditions by the adenosine cyclic 3',5'-phosphate (cAMP)-dependent and/or the calmodulin-dependent protein kinase. The small shifts in apparent molecular weight resulting from the incorporation of Pi groups in the PLB complexes were analyzed by high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In parallel experiments, PLB was dissociated into its subunits and analyzed by using a newly developed isoelectric focusing system. The pI values of the PLB subunits phosphorylated by the cAMP- or calmodulin-dependent kinase were 6.2 and 6.4, respectively. Double phosphorylation of the same subunit resulted in an acidic shift of the pI to 5.2. The combined analysis of the behavior of the PLB complex and of its subunits has greatly simplified the interpretation of the complex phosphorylation pattern and has led to the following conclusions: The PLB complex is composed of five probably identical subunits, each of them containing a distinct phosphorylation site for the calmodulin- and the cAMP-dependent kinase. The population of PLB interacting with the endogenous calmodulin-dependent kinase cannot be phosphorylated by the cAMP-dependent kinase unless previously phosphorylated in the presence of calmodulin. It was also observed that after maximal phosphorylation of PLB in the presence of very large amounts of the cAMP-dependent protein kinase, the Ca2+ pumping rate of the cardiac SR ATPase is stimulated up to 5-fold, i.e., a level of a stimulation which exceeds considerably the values so far reported in the literature.     

Association of type II cAMP-dependent protein kinase with p34cdc2 protein kinase in human fibroblasts

Previous independent studies suggested that type II cAMP-dependent protein kinase and the p34cdc2 protein kinase cell cycle regulator co-localize at centrosomes. In order to investigate whether there is an association of type II cAMP-dependent protein kinase with p34cdc2 in human fibroblasts, we used three different approaches. First, the regulatory subunits RI and RII were photoaffinity-labeled with 8-N3-[32P]cAMP, and anti-p34cdc2 immunoprecipitates were screened for the presence of either RI or RII regulatory subunits by one- or two-dimensional gel electrophoresis. Second, anti-RII alpha immunoprecipitates were screened for the presence of p34cdc2 by Western blot using three different affinity-purified antibodies recognizing different domains of human p34cdc2. Conversely, anti-p34cdc2 immunoprecipitates (three different antibodies), as well as the material retained on p13suc1-Sepharose Bio-Beads, which binds specifically p34cdc2, were screened for the presence of RII alpha. Finally, we have looked for cAMP-dependent protein kinase activity specifically inhibited by PKI in immunoprecipitates obtained from extracts treated with different anti-p34cdc2 antibodies. All these experiments gave concordant results and demonstrate that at least at G0/G1, human fibroblasts contain a complex of active type II cAMP-dependent protein kinase associated through its RII alpha subunit with p34cdc2.     

Phosphorylation modulates keratin structure.

Bovine hoof keratin was shown to be a substrate for cAMP-dependent protein kinase using [gamma-32P]ATP. Natural-abundance cross-polarization (CP) MAS 13C NMR was used to examine the effect of phosphorylation on keratin structure. When short contact times were used, phosphorylation was shown to increase the number of residues in the motionally restricted portions of the protein; i.e., a portion(s) of the protein became more rigid upon phosphorylation. Circular dichroism (CD) spectra showed a spectral shape characteristic of alpha helix for this keratin. Phosphorylation of the keratin by cAMP-dependent protein kinase resulted in a CD spectrum with the same shape but of greater apparent intensity. This may have been the result of an increase in the alpha-helical content of the protein. These data showed that the structure of keratin changed significantly upon phosphorylation by cAMP-dependent protein kinase. The region of the keratin molecule most likely to be altering its structure was the end of the molecule, which was involved in the formation of, and intracellular attachment of, intermediate filaments. Therefore, these data suggested that cAMP-dependent phosphorylation may produce significant changes in the intracellular organization of intermediate filaments. When the keratin was phosphorylated using cold ATP, magic-angle spinning (MAS) 31P nuclear magnetic resonance (NMR) revealed two resonances arising from the phosphorylation sites on the keratin. The more shielded resonance was shown to arise from cAMP-dependent protein kinase phosphorylation. Static 31P NMR measurements suggested that at least two classes of cAMP-dependent sites existed with the same isotropic 31P chemical shift; one was considerably motionally restricted with respect to the other.     

Induction of cAMP-dependent protein kinase I during human monocyte differentiation

Differentiation of human peripheral blood monocytes into macrophages was accompanied by induction of the regulatory subunit of cAMP-dependent protein kinase I as determined by photoaffinity labeling of cytosol proteins with 8-N3-[32P]cAMP and DEAE-Sephacel chromatography. The appearance of cAMP-dependent protein kinase I in macrophages was not due to translocation from the particulate fraction of monocytes. The regulatory subunit of cAMP-dependent protein kinase II was present in both monocytes and in vitro-differentiated macrophages. Protein kinase I in macrophages demonstrated higher affinity for 8-N3-cAMP (KD = 0.7 nM) than did protein kinase II from either monocytes (KD = 14.5 nM) or macrophages (KD = 4.9 nM). These studies demonstrate induction of the regulatory subunit of cAMP-dependent protein kinase I during the differentiation of a normal human cell and support the hypothesis that cAMP may regulate some stages of differentiation.     

Protein phosphorylation and the two stages of pigment organelle dispersion in permeabilized xanthophores: organelle protein phosphorylation alone supports only the first stage

We reported previously that, in cultured goldfish xanthophores, dispersion of aggregated carotenoid droplets (CDs) requires the specific phosphorylation of the CD protein p57 by a cAMP-dependent protein kinase and the presence of cytosol. We report here that, in permeabilized cells, the addition of the catalytic subunit of cAMP-dependent protein kinase and ATP phosphorylates p57 and converts the CDs from an immobile to a mobile state (first stage of CD dispersion). However, the CDs are restricted to the vicinity of the original site of the CD aggregate and do not actually disperse (second stage of CD dispersion) unless cytosol is also added. We propose that this process may be related to aspects of secretory processes.     

Phosphorylation of smooth muscle myosin light chain kinase by protein kinase C. Comparative study of the phosphorylated sites

Smooth muscle myosin light chain kinase is phosphorylated in vitro by protein kinase C purified from human platelets. When myosin light chain kinase which has calmodulin bound is phosphorylated by protein kinase C, 0.8-1.1 mol of phosphate is incorporated per mol of myosin light chain kinase with no effect on its enzyme activity. Phosphorylation of myosin light chain kinase with no calmodulin bound results in the incorporation of 2-2.4 mol of phosphate and significantly decreases the rate of myosin light chain kinase activity. The decrease in myosin light chain kinase activity is due to a 3.3-fold increase in the concentration of calmodulin necessary for the half-maximal activation of myosin light chain kinase. The sites phosphorylated by protein kinase C and the catalytic subunit of cAMP-dependent protein kinase were compared by two-dimensional peptide mapping following extensive tryptic digestion of phosphorylated myosin light chain kinase. The single site phosphorylated by protein kinase C when calmodulin is bound to myosin light chain kinase (site 3) is different from that phosphorylated by the catalytic subunit of cAMP-dependent protein kinase (site 1). The additional site that is phosphorylated by protein kinase C when calmodulin is not bound appears to be the same site phosphorylated by the catalytic subunit of cAMP-dependent protein kinase (site 2). These studies confirm the important role of site 2 in binding calmodulin to myosin light chain kinase. Sequential studies using both protein kinase C and the catalytic subunit of cAMP-dependent protein kinase suggest that the phosphorylation of site 1 also plays a part in decreasing the affinity of myosin light chain kinase for calmodulin.     

Increased level of cAMP-dependent protein kinase in aging human lung fibroblasts

Regulation of the expression of cAMP-dependent protein kinase in cellular aging was studied using the IMR-90 diploid human lung fibroblasts. The level of cAMP-dependent protein kinase present in cell extracts was monitored by 1) photoactivated incorporation of 8-N3-[32P]cAMP into the 47,000- and 54,000-dalton regulatory subunits of the type I and type II cAMP-dependent protein kinases, respectively; 2) cAMP-dependent phosphorylation of histone II AS catalyzed by the catalytic subunit of the kinase; and 3) fractionation and analysis of the type I and type II cAMP-dependent protein kinase by DEAE-Sephacel column chromatography. Our results showed an approximately two- to threefold increase in the level of the type I cAMP-dependent protein kinase and a somewhat smaller increase in the type II kinase in extracts of the "old" IMR-90 cells (population doubling greater than 48) as compared to that of the "young" cells (PDL 22-27). The timing of the increase in cAMP-dependent protein kinase coincided with a significant decrease in the proliferative potential of the cells. This result together with previously demonstrated effects of cAMP in the control of cell growth and differentiation and the increased expression of cAMP-dependent protein kinase during terminal differentiation of the murine preadipocytes (3T3-L1) and myoblast (L-5, L-6, and C2C13) suggests that regulation of the levels of cAMP and cAMP-dependent protein kinase plays a significant role in the control of cell growth and differentiation.     

Activators of protein kinase C up-regulate the cell surface expression of CD2 and CD5 T cell glycoproteins

Activation of human T cells through the CD3-T cell receptor complex caused an augmentation in the cell surface expression of CD2 and CD5 glycoproteins. Evidence that protein kinase C is involved in the up-regulatory mechanism of these cell surface molecules has been obtained by three different approaches: (a) the changes in antigen expression were observed with activators of protein kinase C such as phorbol esters but not with activators of kinases dependent on calcium/calmodulin or cAMP; (b) the overexpression of CD2 and CD5 is also observed in cells treated with 1,2-dioctanoyl-rac-glycerol, an analogue of the physiological activator of protein kinase C; and (c) 1-(5-isoquinolinyl)-2-methylpiperazine, an inhibitor of protein kinase C but not N-(2-guanidinoethyl)-5-isoquinolinesulfonamide dihydrochloride, an inhibitor of the cAMP-dependent kinase, impairs CD2 and CD5 up-regulation. These changes in cell surface antigen expression appear to be caused by the concomitant increase in the mRNA levels for CD2 and CD5. Phosphorylation studies of the CD2 and CD5 glycoproteins indicated that the overexpression of these molecules was not associated with a specific pattern of phosphorylation since it was observed independently of their hyperphosphorylated or nonphosphorylated state.     

Characterization of cyclic AMP-requiring yeast mutants altered in the regulatory subunit of protein kinase

The CYR3 mutant of yeast, Saccharomyces cerevisiae, partially accumulated unbudded cells and required cAMP for the best growth at 35 degrees C. The CYR3 mutation was partially dominant over the wild type counterpart and suppressed by the bcy1 mutation which is responsible for the deficiency of the regulatory subunit of cAMP-dependent protein kinase. The molecular weights of cAMP-dependent protein kinase and its catalytic and regulatory subunits were 160,000, 30,000, and 50,000, respectively. No significant differences in the molecular weights of cAMP-dependent protein kinase and the subunits were found between the wild type and CYR3 mutant strains. However, the cAMP-dependent protein kinase activity of CYR3 cells showed significantly higher Ka values for activation by cAMP at 35 degrees C than those of wild type and a clear difference in the electrophoretic mobility of the regulatory subunit was found between the wild type and CYR3 enzymes. The CYR3 mutation was suppressed by the IAC mutation which caused the production of a significantly high level of cAMP. The results indicate that the CYR3 phenotype was produced by a structural mutation in the CYR3 gene coding for the regulatory subunit of cAMP-dependent protein kinase in yeast.     

Evidence that cAMP-dependent protein kinase and a protein factor are involved in reactivation of triton X-100 models of sea urchin and starfish spermatozoa

A fraction obtained from detergent-extract of sea urchin or starfish spermatozoa using DEAE-cellulose chromatography reactivated Triton X-100 models of the spermatozoa in a cAMP-dependent manner. The DEAE fraction contained cAMP-dependent protein kinase with a high level of specific activity. Rabbit muscle inhibitor protein highly specific for cAMP-dependent protein kinases inhibited the ability of the deae fraction to induce reactivation of Triton X-100 models.l This inhibition paralleled inhibition of cAMP-dependent protein kinase activity of the DEAE fraction, suggesting participation of the enzyme in the cAMP-dependent reactivation of Triton X-100 models. However, cAMP-dependent protein kinase further purified from the DEAE fraction was incapable of reactivating these models by itself. A protein factor which was separated from the protein kinase in the course of purification of the enzyme was found to also be necessary for the reactivation. When cAMP-dependent protein kinase was pretreated with protein kinase inhibitor before addition of the protein factor, the reactivation of Triton X-100 models was no longer detected. However, after the protein factor had been incubated with cAMP and cAMP-dependent protein kinase, protein kinase inhibitor did not repress reactivation of Triton X-100 models. We propose that the reactivation needs phosphorylation of the protein factor by cAMP-dependent protein kinase.     

PKIB抑制PKA活性调节细胞衰老

人cAMP依赖型蛋白激酶B抑制剂(PKIB)是一种新的耐热蛋白.实验证明,PKIB对PKA催化亚单位具有抑制作用.为研究PKIB在细胞衰老中的功能,以年轻和年老人胚肺二倍体成纤维细胞(2BS细胞株)为实验对象,通过实时 PCR发现,在年老细胞中,PKIB表达高于年轻细胞;用PKA活化剂处理年轻和年老细胞,结果显示,在年老细胞中PKA活性变化较年轻细胞小.通过免疫共沉淀实验证实,在年老细胞中,PKIB与PKA催化亚单位结合较年轻细胞中多;进一步通过在年轻细胞中过表达PKIB,检测细胞PKA活性,发现PKA活性明显降低,进一步证实了在人胚肺二倍体成纤维细胞中PKIB对PKA活性的抑制作用;利用Western实验结果证实,PKA催化亚单位在年轻细胞中的表达高于年老细胞.以上结果证明,在2BS细胞中,PKA活性受PKIB的抑制;这种抑制作用与细胞的衰老有一定的关联作用.     

Effects of phosphorylation of the neurofilament L protein on filamentous structures.

Effects of phosphorylation of the neurofilament L protein (NF-L) on the reassembly system were studied by both sedimentation experiments and low-angle rotary shadowing. Bovine spinal cord NF-L was phosphorylated with 3-4 mol/mol protein by either the catalytic subunit of cAMP-dependent protein kinase or protein kinase C. Phosphorylated NF-L could not assemble into filaments. Phosphorylation by either cAMP-dependent protein kinase or protein kinase C inhibited the same step of the reassembly process. Phosphorylated NF-L remained as an 8-chain complex even in favorable conditions for reassembly. The extent of the effect of phosphorylation on the filamentous structure of NF-L was also investigated by using the catalytic subunit of cAMP-dependent protein kinase. The amount of unassembled NF-L increased linearly with increased phosphorylation in the sedimentation experiments. Structural observations indicated that 1 or 2 mol of phosphorylation is enough to inhibit reassembly and to induce disassembly, and the disassembly process was also observed. The filaments were shown to unravel with disassembly. Star-like clusters, which we reported as being the initial stage of reassembly, were also identified.     

Regulation of the interaction of actin filaments with microtubule-associated protein 2 by calmodulin-dependent protein kinase II

Phosphorylation of microtubule-associated protein 2 (MAP 2) by Ca²⁺-, calmodulin-dependent protein kinase II (protein kinase II) inhibited the actin filament cross-linking activity of MAP 2. This inhibition required the presence of ATP, Mg²⁺, Ca²⁺ and calmodulin. The minimal concentration of MAP 2 required for gel formation of actin filaments was increased with increasing amounts of phosphate incorporated into MAP 2, and the phosphorylated MAP 2, into which 10.3 mol of phosphate/mol of protein had been incorporated, did not cause actin filaments to gel under the experimental conditions used. The phosphorylation of MAP 2 by Ca²⁺-, phospholipid-dependent protein kinase (protein kinase C) and cAMP-dependent protein kinase also inhibited the actin filament cross-linking activity of MAP 2. The extent and rate of phosphorylation of MAP 2 by protein kinase II were higher than those of the phosphorylation by protein kinase C and cAMP-dependent protein kinase. The interaction of actin filaments with MAP 2 was inhibited more by the actions of protein kinase II and protein kinase C than by cAMP-dependent protein kinase. The actin filament cross-linking activity of MAP 2 phosphorylated either by protein kinase II, cAMP-dependent protein kinase or protein kinase C was retrieved when phosphorylated MAP 2 was treated by protein phosphatase. These results indicate that the interaction of actin filaments with MAP 2 is regulated by the phosphorylation-dephosphorylation of MAP 2.     

Expression of a mutant regulatory subunit of cAMP-dependent protein kinase in the Caco-2 human colonic carcinoma cell line.

Caco-2 human colonic carcinoma cells were transfected with an expression vector encoding a mutant form of RI (regulatory subunit of the type 1 cAMP-dependent protein kinase), driven by the metallothionein 1 promoter. A stable transformant was isolated that expressed the mutant RI gene in a Zn(2+)-inducible manner. The consequences of the RI mutation on cAMP-dependent protein kinase activity, cell division, and regulation of chloride efflux were examined. When grown in the absence of ZnSO4, protein kinase activity in the transformant was stimulated 2.5-fold by cAMP and approached the levels of cAMP-dependent protein kinase activity seen in parental Caco-2 cells; when treated with ZnSO4, cAMP-dependent protein kinase activity in the transformant was inhibited by 60%. In the absence of ZnSO4 the transformant grew with the same doubling time and to the same saturation density as the untransformed parent. In the presence of ZnSO4 the transformant exhibited a cAMP-reversible inhibition of cell division, indicating that a functional cAMP-dependent protein kinase was required for the growth of these cells in culture. Induction of the mutant RI gene also abolished forskolin-stimulated chloride efflux from these cells, suggesting obligatory roles for cAMP and cAMP-dependent protein kinase in forskolin's actions on chloride channel activity. We anticipate that this transformant will be useful for further studies on the roles of cAMP and cAMP-dependent protein kinase in the regulation of intestinal epithelial cells, including regulation of cell proliferation and differentiation, and regulation of chloride channel activity by neurohormones and neurotransmitters.     

PrKX is a novel catalytic subunit of the cAMP-dependent protein kinase regulated by the regulatory subunit type I

The human X chromosome-encoded protein kinase X (PrKX) belongs to the family of cAMP-dependent protein kinases. The catalytically active recombinant enzyme expressed in COS cells phosphorylates the heptapeptide Kemptide (LRRASLG) with a specific activity of 1.5 micromol/(min.mg). Using surface plasmon resonance, high affinity interactions were demonstrated with the regulatory subunit type I (RIalpha) of cAMP-dependent protein kinase (KD = 10 nM) and the heat-stable protein kinase inhibitor (KD = 15 nM), but not with the type II regulatory subunit (RIIalpha, KD = 2.3 microM) under physiological conditions. Kemptide and autophosphorylation activities of PrKX are strongly inhibited by the RIalpha subunit and by protein kinase inhibitor in vitro, but only weakly by the RIIalpha subunit. The inhibition by the RIalpha subunit is reversed by addition of nanomolar concentrations of cAMP (Ka = 40 nM), thus demonstrating that PrKX is a novel, type I cAMP-dependent protein kinase that is activated at lower cAMP concentrations than the holoenzyme with the Calpha subunit of cAMP-dependent protein kinase. Microinjection data clearly indicate that the type I R subunit but not type II binds to PrKX in vivo, preventing the translocation of PrKX to the nucleus in the absence of cAMP. The RIIalpha subunit is an excellent substrate for PrKX and is phosphorylated in vitro in a cAMP-independent manner. We discuss how PrKX can modulate the cAMP-mediated signal transduction pathway by preferential binding to the RIalpha subunit and by phosphorylating the RIIalpha subunit in the absence of cAMP.     

Characterization of calmodulin-activated protein kinase activity of rat adipocyte endoplasmic reticulum fraction

Calmodulin-activated protein kinase activity in the endoplasmic reticulum fraction of rat adipocytes was identified and characterized. The major endogenous protein substrate of the calmodulin-activated kinase activity has an apparent molecular weight of 54,000 as determined by sodium dodecyl sulfate gel electrophoresis. The calmodulin-activated component of the activity was saturated at 10 microM ATP. Calcium or calmodulin alone did not increase the activity, but the simultaneous presence of calcium and calmodulin increased activity three to four-fold. Half-maximal activation of this activity occurred at 8 microM Ca2+. The addition of increasing amounts of calmodulin caused a concentration-dependent activation in the presence of calcium, which was saturable at high calmodulin concentrations. Magnesium was required for activity, with half-maximal activity occurring at 230 microM. The antipsychotic drug trifluoperazine inhibited the activation of the protein kinase activity by calmodulin, but had a negligible effect on the basal activity. Half-maximal inhibition occurred at 63 microM. Phosphorylation of the 54,000 mol. wt band was independent of cAMP, cGMP and the combination of cAMP and cAMP-dependent protein kinase. Calmodulin-activated protein kinase phosphorylated both phosphoserine and phosphothreonine residues in the 54,000 mol. wt substrate. These experiments have partially characterized a calmodulin-activated protein kinase activity from adipocytes, which appears to be a unique activity of unknown function.     

cAMP-dependent protein kinase stimulates epidermal growth factor-dependent phosphorylation of epidermal growth factor receptors

Epidermal growth factor (EGF)-dependent transfer of radiolabeled phosphate from [gamma-32P]ATP to 160-kDa EGF receptor solubilized from human epidermoid carcinoma A431 cell surface membranes was stimulated up to 3-fold by addition of 3',5'-cAMP and purified cAMP-dependent protein kinase. Phosphorylation of EGF receptors was stimulated to the same extent when cAMP-dependent protein kinase catalytic subunit was substituted for 3',5'-cAMP and cAMP-dependent protein kinase. Phosphoamino acid analysis revealed that the extent of phosphorylation of EGF receptor at tyrosine residues was the same regardless of whether cAMP-dependent protein kinase catalytic subunit was present in or omitted from the system. Increased EGF receptor phosphorylation occurring in response to cAMP-dependent protein kinase catalytic subunit was accounted for by phosphorylation at serine or threonine residues. In samples phosphorylated in the presence of cAMP-dependent protein kinase catalytic subunit, phosphate was present in tyrosine, serine, and threonine in a ratio of 32:60:8. Two-dimensional mapping of radiolabeled phosphopeptides produced from EGF receptors by digestion with trypsin revealed the generation of one additional major phosphoserine-containing peptide when cAMP-dependent protein kinase was present with EGF in the EGF receptor kinase system. Degradation of 160-kDa EGF receptors to a 145-kDa form by purified Ca2+-activated neutral protease produced a 145-kDa fragment with phosphoserine content increased over that present initially in the 160-kDa precursor.