Nuclear protein synthesis and phosphorylation in Friend erythroleukemia cells stimulated with DMSO

Patterns of nuclear protein synthesis and phosphorylation have been investigated in Friend erythroleukemia cells. The rate of incorporation of [³H]leucine and [³²P]phosphate remains relatively constant during the first 48 h of dimethylsulfoxide (DMSO) stimulation, when more than 90% of the cells commit to erythroid differentiation, but falls to 20% by 120 h. Histone H2A phosphorylation is greatly increased during DMSO treatment, but no significant changes were found in the non-histone phosphoprotein patterns as determined by gel electrophoresis. There is also a small, but reproducible, change in the relative amounts of the two sub-fractions of histone H2A. There are no striking changes in the electrophoretic patterns of [¹⁴C]leucine-labelled nuclear proteins during the first 48 h, but the amount and the synthesis of two proteins of 46 000 and 280 000 D are increased somewhat during this period. Another protein, of molecular weight 65 000, appears to be induced in low amounts.     

The effect of cyclic nucleotides and cholera toxin on in vivo and in vitro phosphorylation of small intestinal brush border membranes

The effect of cyclic nucleotides and cholera toxin on the phosphorylation of the brush border membrane proteins of the rat jejunum was studied. Phosphorylation was analyzed by autoradiography of brush border membrane proteins separated by SDS-polyacrylamide gel electrophoresis. Phosphorylation was performed either in vivo by perfusion of the jejunum with [³²P]orthophosphate followed by an analysis of the isolated membranes or in vitro by phosphorylation of isolated brush border membranes by [γ-³²P]ATP in the presence of saponin. The addition of cholera toxin (10 μg/ml) or dibutyryl-cAMP (5 mmol/l) to the perfusate was unable to produce significant changes in the phosphoprotein pattern. On the other hand, cAMP (at 5 μmol/l) induced an increase of the phosphorylation of a 86 kDa protein when freshly isolated brush border membranes were phosphorylated by [γ-³²P]ATP. However, the same effect could also be induced by low concentrations of cGMP (0.1 μmol/l). It is concluded that brush border membranes from rat jejunum do not contain cAMP-dependent protein kinase activity and that cAMP-dependent protein phosphorylation of this membrane does probably not represent the final event of cholera toxin-induced secretion.     

Phosphorylation of a nucleolus-specific phosphoprotein in vitro.

The cellular site and characteristics of the phosphorylation of a nucleolus-specific phosphoprotein (molecular weight, 120 000) in mouse ascites tumor cells were studied. The phosphoprotein was strongly labeled with ³²P when the isolated nucleoli were incubated with [γ-³²P]ATP in vitro. This phosphoprotein, and protein kinase for the protein phosphorylation were both purified from 0.3 M KCl soluble protein fraction of the nucleoli by hydroxylapatite and phosphocellulose column chromatographies. It was found that phosphorylation of the nucleolus-specific phosphoprotein was catalyzed selectively by a guanosine 3:5-monophosphate-dependent protein kinase in the nucleoli and the reaction product was the same phosphoprotein as the substrate used.     

Phosphorylation of Thylakoid Proteins of Oryza sativa: In Vitro Characterization and Effects of Chilling Temperatures

The phosphorylation of thylakoid proteins of rice (Oryza sativa L.) was studied in vitro using [γ-³²P]ATP. Several thylakoid proteins are labeled, including the light-harvesting complex of photosystem II. Protein phosphorylation is sensitive to temperature, pH, and ADP, ATP, and divalent cation concentrations. In the range pH 7 to 8.2, phosphorylation of the light-harvesting polypeptides declines above pH 7.5, whereas labeling of several other thylakoid polypeptides increases. Increasing divalent cation concentration from 3 to 20 millimolar results in a decrease in phosphorylation of the 26 kilodalton light-harvesting complex polypeptide and increased phosphorylation of several other polypeptides. ADP has an inhibitory effect on the phosphorylation of the light-harvesting complex polypeptides. Phosphorylation of the 26 kilodalton light-harvesting polypeptide requires 0.45 millimolar ATP for half-maximal phosphorylation, compared to 0.3 millimolar for the 32 kilodalton phosphoprotein. Low temperature inhibits the phosphorylation of thylakoid proteins in chilling-sensitive rice. However, phosphorylation of histones by thylakoid-bound kinase(s) is independent of temperature in the range of 25 to 5°C, suggesting that the effect of low temperature is on accessibility of the substrate, rather than on the activity of the kinase.     

Effect of parathyroid hormone and cyclic AMP on protein phosphorylation in rabbit kidney cortex

Suspensions of renal cortical tubules were incubated with ³³P_i and exposed to parathyroid hormone (40 μg/ml) or 1 mM dibutyryl cyclic AMP. In other experiments homogenates of renal cortex were assayed for protein kinase and phosphoprotein phosphatase activity using [γ-³²P]ATP with or without 5 mM cyclic AMP. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and phosphorylation of proteins measured by liquid scintillation counting of gel slices. The pattern of protein phosphorylation was similar in control tissue from both tubule suspensions and homogenates. In intact tubules, parathyroid hormone stimulated the phosphorylation of four proteins with molecular weights of approx. 1500 000, 125 000, 100 000 and 50 000 by 28%, 24%, 13%, and 20%, respectively. Results with dibutyryl cyclic AMP were comparable but more variable. Stimulation of phosphorylation by cyclic AMP in homogenates was more generalized with the major effect on a 50 000 dalton protein (50% stimulation). No effect of cyclic AMP on dephosphorylation of proteins was observed. The results are interpreted as indicating that increased phosphorylation of cell proteins is part of the cyclic AMP-mediated response of the renal cortex to parathyroid hormone.     

Partial characterization of protein kinase-catalyzed phosphorylation of low molecular weight proteins in purified preparations of pigeon heart sarcolemma and sarcoplasmic reticulum

Pigeon heart microsomes contain three minor size protein kinase substrates of minimal molecular weights of 22 000, 15 000, and 11 500, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When the microsomes were partially loaded with calcium oxalate and subjected to rate zonal and isopynic centrifugations in sucrose density gradient columns, the 22 000 and the 15 000 dalton proteins settled in the heaviest fraction, which was composed mainly of vesicles of sarcoplasmic reticular membranes; the 11 500 dalton protein was concentrated in the lightest fractions, which consisted chiefly of vesicles of sarcolemmal origin. During incubation of the membrane fractions with Mg[γ-³²P]ATP significant amounts of ³²P were incorporated into all these proteins. Incorporation of ³²P into the 15 000 dalton protein was moderately and ³²P incorporation into the 22 000 dalton protein was markedly enhanced in the presence of exogenous soluble cyclic AMP-dependent protein kinase and cyclic AMP. The phosphorylation of the three proteins was virtually unaffected by CA²⁺ concentrations up to 0.1 mM and by ethyleneglycol-bis(β-aminoethylether)-N,N′-tetraacetic acid in the absence of added Ca²⁺.Phosphorylation of the 22 000 and the 11 500 dalton proteins occurred mainly at serine residues. In the 15 000 dalton protein threonine residues were the main site of endogenous phosphorylation. Nearly equal amounts of [³²P]-phosphate were incorporated into threonine and serine residues of this protein when phosphorylation was supported by exogenous cyclic AMP-dependent protein kinase and cyclic AMP.The 15 000 dalton protein could be removed from its membrane attachment by extraction with an acidic chloroform/methanol mixture. This step opens the way for the purification of this membrane-bound protein kinase substrate.     

Endogenous hyperphosphorylation in plasma membrane from an ascites hepatocarcinoma cell line

Plasma-membrane-bound kinases of AS-30D ascites from transplantable rat hepatocarcinoma were shown to extensively catalyze the phosphorylation of plasma membrane proteins and membrane lipids, using [gamma-32P]ATP or [gamma-32P]GTP as a phosphate donor. In contrast, plasma membranes from normal adult rat liver or fast-growing regenerating liver (24 h after partial hepatectomy) produce significantly less activity for protein phosphorylation and little phosphorylation of the lipids. However, neonatal (24 h old) rat liver plasma membrane preparations show levels of phosphorylation of proteins and lipids intermediate between those in the tumor cell line and normal adult plasma membrane preparations. Phosphatidic acid was identified as one of the 32P-labelled lipids in the tumor plasma membrane chloroform-methanol (2:1, v/v) extract. Phosphorylation of protein was not affected by cAMP or cGMP. However, calcium ion (in the presence or absence of calmodulin) significantly modifies the 32P labelling of a series of proteins in normal tissue but has little effect with the neoplastic preparations. Some plasma membrane proteins were capable of nucleotide binding, instead or in addition to being phosphorylated. Finally, the presence of membrane-bound phosphoprotein phosphatase(s) was also demonstrated in all the preparations examined by means of chase experiments with nonlabelled ATP or GTP, and (or) by the use of the phosphoprotein phosphatase inhibitor, orthovanadate.     

Cyclic nucleotide- and calcium-independent phosphorylation of proteins in rat brain polyribosome: Effects of ACTH,spermine, and hemin

The incorporation of [-³²P]ATP into proteins of rat brain polyribosomes was studied in vitro. The effects of cyclic nucleotides, calcium, hemin, ACTH, GTP, and spermine were examined. The incorporation of phosphate into proteins increased with time and phosphatase activity was very low; thus, the extent of phosphorylation was predominantly a reflection of protein kinase activity. Phosphorylation of proteins was not sensitive to Ca²⁺ in the presence or absence of either calmodulin or phosphatidylserine. Phosphorylation was also unaffected by cyclic nucleotides in the absence of exogenous enzymes. However, addition of a cMAP-dependent protein kinase together with cAMP resulted in a stimulation of the incorporation of phosphate into 4 phosphoproteins (pp70, pp58, pp43, and pp32); phosphorylation of pp32 was completely dependent on the addition of the kinase. ACTH (1–24), (11–24), and spermine inhibited the endogenous phosphorylation of one protein band (pp30). The phosphorylation of this 30 kD band was also selectively increased by hemin (5 M). Higher concentrations of hemin exerted an inhibitory effect on the majority of the phosphoproteins. Protein phosphatase activity was not influenced by ACTH or spermine. The specific inhibition of pp30 phosphorylation by ACTH or spermine is most probably explained by an interaction with a cyclic nucleotide- and Ca²⁺-independent protein kinase.     

Phosphorylation of the IgE receptor from ionophore A23187 stimulated intact rat mast cells

Purified rat serosal mast cells were labeled either with [³²P]orthophosphate or [³⁵S]methionine and their receptors for immunoglobulin E were isolated by repetitive affinity chromatography. In ³⁵S-labeled receptor preparations SDS polyacrylamide gels revealed a broad receptor band, M_r 45,000 to 53,000, and two other bands, M_r 30,000 and 16,000, which apparently represent receptor-associated proteins. Only the receptor band was labeled by ³²P. Phosphorylation of receptor was markedly stimulated by the divalent cation ionophore A23187, a known stimulator of histamine release, with changes occuring as early as 15 seconds. This early increase in receptor phosphorylation may be involved in the control of mediator secretion.     

Characterization of specific differences in protein phosphorylation of the plasma membrane and the endoplasmic reticulum of mouse fibroblasts

Endogenous phosphorylation was studied with highly purified fractions of the plasma membrane and the endoplasmic reticulum of SV40-transformed mouse fibroblasts using [γ-³²P]ATP and [γ-³²P]GTP as precursors. With ATP maximum overall incorporation of ³²P into both membrane fractions occured at pH 7.8 in the presence of 10 mM MgCl₂ after incubation for 1 min. GTP could be utilized only by the plasma membrane fraction showing maximum incorporation of ³²P at pH 7.8 and 10 mM MgCl₂ after incubation for 3 min.The pattern of phosphoproteins of the plasma membrane is represented by more than 15 proteins whereas the endoplasmic reticulum essentially contained only one phosphorylated component of 35 000 molecular weight. The comparison of ATP- and GTP-specific phophorlation of the plasma membrane revealed GTP to be a less efficient precursor yielding a similar phosphoprotein pattern with one significant difference: the GTP-specific main component exhibited a molecular wieght of about 100 000 and the ATP-specific main component a molecular weight of 110 000.The relative distribution of individual phosphoproteins in the pattern of the plasma membrane was dependent on pH but not on MgCl₂ concentration or time of incubation. Increasing concentrations of plasma membrane protein altered the patterns of phosphoproteins dramatically: At high protein concentrations the ATP-specific main component (M_r = 110 000) was no more phosphorylated whereas with GTP the main component M_r = 100 000 was essentially the sole phosphorylated protein.     

Acidic-phosphoprotein phosphatase activity of rat ventral prostate nuclei apparent lack of effect of androgens

A protein phosphatase activity has been demonstrated in nuclei of rat ventral prostate utilizing ³²P-labelled phosvitin as a model acidic phosphoprotein substrate. This phosphoprotein phosphatase has a pH optimum of 6.7, is unaffected by the sulphydryl protecting agent 2-mercaptoethanol, and requires a divalent cation for maximal activity. Of the various divalent cations tested, Mg²⁺ is the most effective in reactivating the EDTA-inhibited enzyme. The phosphatase is inhibited by sodium fluoride, sodium oxalate, N-ethylmaleimide, ATP and ADP but is relatively insensitive to ammonium molybdate. Increased ionic strength of the reaction medium also causes a reduction in the enzyme activity, e.g., by 48% at 200 mM sodium chloride. The activity of the acidic phosphoprotein phosphatase did not change significantly at 48 h or 96 h postorchiectomy when expressed per unit of nuclear protein. However, it is reduced by approx. 30% at these times after castration if based on DNA content. The decline in activity per nucleus reflects the decrease in the realtive nuclear protein content observed at 48 h or 96 h post-orchiectomy. This suggests that the decline in the phosphorylation of prostatic nuclear acidic proteins which occurs upon androgen withdrawal is not due to increased nuclear phosphatase activity.     

Phosphorylation of muscle plasma membrane protein by a membrane-bound protein kinase

Protein kinase activity has been demonstrated in purified plasma membranes from rat diaphragm by measuring the incorporation of ³²P from [³²P]-ATP into endogenous membrane proteins and into histone, in vitro. Histone appears to be a better substrate than the endogenous membrane proteins; however, the properties of the enzyme are similar when phosphorylating endogenous or exogenous proteins. The activity of this membrane-associated protein kinase is not significantly affected by cyclic adenosine 3′,5′-monophosphate or by cyclic guanosine 3′,5′-monophosphate, but is inhibited by theophylline. The ³²P incorporated into membrane proteins is alkali-labile and is released from the membrane by protease digestion, but it is not removed by phospholipase C, by hydroxylamine, or by chloroform—methanoll extraction. Solubilization of ³²P-labeled membranes by sodium dodecylsulfate and fractionation by sodium dodecylsulfate polyacrylamide gel electrophoresis reveals that the radioactivity is predominantly associated with a single protein band with an apparent molecular weight of about 51 000. The phosphoprotein is a minor membrane component as judged by Coomassie blue staining.     

In vitro and in vivo phosphorylation of polypeptides in plasma membrane and tonoplast-enriched fractions from barley roots

Phosphorylation of polypeptides in membrane fractions from barley (Hordeum vulgare L. cv CM 72) roots was compared in in vitro and in vivo assays to assess the potential role of protein kinases in modification of membrane transport. Membrane fractions enriched in endoplasmic reticulum, tonoplast, and plasma membrane were isolated using sucrose gradients and the membrane polypeptides separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis. When the membrane fractions were incubated with γ-[³²P]ATP, phosphorylation occurred almost exclusively in the plasma membrane fraction. Phosphorylation of a band at 38 kilodaltons increased as the concentration of Mg²⁺ was decreased from millimolar to micromolar levels. Phosphorylation of bands at 125, 86, 58, 46, and 28 kilodaltons required millimolar Mg²⁺ concentrations and was greatly enhanced by Ca²⁺. When roots of intact plants were labeled with [³²P]orthophosphate, polypeptides at approximately 135, 116, 90, 46 to 53, 32, 28, and 19 kilodaltons were labeled in the plasma membrane fraction and polypeptides at approximately 73, 66, and 48 kilodaltons were labeled in the tonoplast fraction. Treatment of the roots of intact plants with 150 millimolar NaCl resulted in increased phosphorylation of some polypeptides while treatment with 100 mm NaCl had no effect.     

Effects of Ca2+, calmodulin and cyclic AMP on the phosphorylation of endogenous proteins by homogenates of rat islets of Langerhans

Activation of Ca²⁺-calmodulin- and cyclic AMP-dependent protein kinases has been suggested to be involved in stimulus-secretion coupling in the pancreatic β-cell. To study the properties of such kinases and their endogenous protein substrates homogenates of rat islets of Langerhans were incubated with [γ-³²P]ATP. Phosphorylated proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and detected by autoradiography. The phosphorylation of certain proteins could be enhanced by Ca²⁺ plus calmodulin or by cyclic AMP. The major effect of Ca²⁺ and calmodulin was to stimulate the phosphorylation of a protein (P53) of molecular weight 53 100±500 (n = 15). Maximum phosphorylation of protein P53 occurred within 2 min with 2 μM free Ca²⁺ and 0.7 μM calmodulin. Incorporation of label into protein P53 was inhibited by trifluoperazine or W7 but not by cyclic AMP-dependent protein kinase inhibitor. Phosphorylation of a protein of similar molecular weight could be enhanced to a lesser extent in the absence of Ca²⁺ but in the presence of cyclic AMP and 3-isobutylmethylxanthine: this phosphorylation was blocked by cyclic AMP-dependent protein kinase inhibitor. Cyclic AMP also stimulated incorporation of label into polypeptides of molecular weights 55 000 and 70–80 000. The results are consistent with the hypothesis that protein phosphorylation mechanisms may play a role in the regulation of insulin secretion.     

Protein phosphorylation in rat liver mitochondria

Summary Incubation of rat liver mitochondria in the presence of either [³²P] Pi or ₃₂ ^y -P] ATP resulted in a phosphorylation of four proteins with Mr 50, 47, 44 and 36 kDa, respectively. The endogenous phosphorylation of these proteins in the presence of [³²P] Pi was markedly influenced by the osmolarity of the incubation medium and differentially affected by various effectors of mitochondrial functions, such as Ca²⁺, oligomycin, FCCP, arsenite and dichloroacetate. In particular, the 36 kDa protein, unlike the other proteins, appears to be phosphorylated also by direct incorporation of [³²P], independently of respiratory chain-linked ATP synthesis. The four proteins, located in the mitoplasts, seem to be phosphorylated by diiferent protein kinases, as suggested by the observation that the endogenous phosphorylation of 36 kDa protein resulted selectively increased by addition of exogenous protein kinases, such as casein kinases S and TS. A tentative identification of these phosphorylatable protein is discussed.     

Stimulation of CK2-dependent Grp94 phosphorylation by the nuclear localization signal peptide

The nuclear localization signal sequence (NLS) of SV40 Large T antigen is essential and sufficient for the nuclear translocation of the protein. Phosphorylation often modulates the intracellular distribution of signaling proteins. In this study, we investigated effects of the NLS-peptide of Large T antigen on protein phosphorylation. When crude cell lysates were incubated with [γ-³²P]ATP, phosphorylation of several endogenous substrates with molecular masses of 100, 80, 50, and 45 kDa by an endogenous kinase was stimulated by the addition of the wild type NLS-peptide (CPKKKRKVEDP). The mutated NLS-peptide (CPKTKRKVEDP) and the reversed NLS-peptide (PDEVKRKKKPC) are weak in the nuclear localization activity, and they only weakly stimulated phosphorylation of these substrates. The mobility of the 100 kDa phosphoprotein was indistinguishable with that of an endoplasmic reticulum (ER)-resident molecular chaperone glucose-regulated protein 94 (Grp94) belonging to the Hsp90 family, and purified Grp94 was phosphorylated by a kinase in cell lysates in an NLS-dependent fashion. The 100 kDa protein was identified as Grp94 by immunoprecipitation and reconstitution experiments. Purification of the NLS-dependent Grp94 kinase by sequential biochemical column chromatography steps resulted in isolation of two polypeptides with molecular masses of 42 and 27 kDa, which were identified as α and β subunit of protein kinase CK2, respectively, by western blotting analysis and biochemical characterization. Moreover, effect of an excess amount of GTP and V8 peptide mapping showed that the NLS-dependent Grp94 kinase in the cell lysate is identical with CK2. Surprisingly purified CK2 did phosphorylate Grp94 even without the NLS-peptide, suggesting that an additional suppressive factor is required for NLS-dependent phosphorylation of Grp94 by CK2. We suggest a possible general role for CK2-catalyzed phosphorylation in the regulation of NLS-dependent protein nuclear translocation.     

GTP-sensitive phosphorylation of proteins in a postmitochondrial supernatant from rat brainstem affected by ACTH1–24

The effect of ACTH_1–24 and cyclic nucleotides on the endogenous phosphorylation of proteins from a postmitochondrial supernatant from rat brainstem was investigated in the presence and absence of GTP. Phosphorylation and its modulation by these compounds were studied in vitro by incorporation of labeled phosphate from [-³²P]ATP added to the incubation mixture. Phosphoproteins were subsequently analyzed by autoradiography after one-and two-dimensional separation. Eight ACTH-sensitive phosphoproteins of molecular weights 75 (IEP 4.0), 67, 64, 50 (IEP 4.7), 47 (IEP 4.8), 38, 34, and 24K were found. The effects of ACTH on phosphorylation were mainly inhibitory, and the effected protein bands did not coincide with the phosphoproteins sensitive to cyclic AMP and cyclic GMP. Phosphorylation of those phosphoprotein bands and its ACTH sensitivity appeared to be highly sensitive to GTP. It is suggested that the activity of protein kinases involved in hormone-sensitive phosphorylation in a postmitochondrial rat brainstem fraction is regulated by GTP-dependent mechanisms.     

On the role of protein phosphorylation in the ATP-dependent permeabilization of transformed cells

Incubation of transformed mouse fibroblasts with external ATP in alkaline medium low in divalent cations causes an increase in the permeability of the plasma membrane to nucleotides and other small molecules. Previous suggestions that the phosphorylation of a 44,000 dalton membrane protein is involved in this permeabilization process have been pursued. Fractionation of cells that had been incubated with [γ-³²P] ATP revealed that the labeled 44K phosphoprotein was found in both the membrane and mitochondrial fractions. Incubation of fractions isolated from unlabeled cells with [γ-³²P] ATP resulted in substantial formation of ³²P-44K in the mitochondrial fraction and less incorporation in the membrane fraction. The 44,000 dalton protein was identified as the α-subunit of mitochondrial pyruvate dehydrogenase by partial proteolytic mapping and immunological cross-reactivity with antibodies prepared against bovine pyruvate dehydrogenase. The phosphorylation of this protein in whole cells by externally added ATP is suppressed by inclusion in the incubation medium of carboxyatractyloside (CAT) and EDTA. These substances have no effect on ATP-dependent permeabilization, indicating that the phosphorylation of pyruvate dehydrogenase is not involved in this process.     

Transient formation of a phosphoprotein during autophosphorylation of rat mammary gland golgi vesicles

Incubation with [γ-³²P]ATP of Golgi vesicles, prepared from the mammary tissue of lactating rats, resulted in the phosphorylation of four of the proteins in the preparation which were resolvable by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis. Three of these had electrophoretic properties identical to the three major caseins of rat milk: their phosphorylation was approximately linear with respect to time during the course of the short (1 min) incubations analyzed. The fourth component (M_r,app. 70 000) behaved differently. It was very rapidly phosphorylated to a maximum level within 5 s at 0°C; its ³²P-content declined thereafter, with a for dephosphorylation of approx. 20 s. The extent of ³²P incorporation into this component, measured after incubation for 20 s at 0°C with [γ-³²P]ATP, was sensitive to the concentration of Ca²⁺ in the incubation medium, being enhanced at low concentrations (<10⁻⁸ M) of Ca²⁺ and depressed at high (10⁻⁴ M) ones. Inclusion of ADP (100 μM) in such incubations also depressed ³²P incorporation into the 70 kDa component. This phosphoprotein was further distinguished from the other three by virtue of the lability of its incorporated phosphorus to treatment with hot trichloroacetic acid. The properties and possible function of this phosphoprotein are discussed in relation to the ATP-dependent Ca²⁺ transport that occurs in this Golgi vesicle preparation.     

Phosphorylation of the contact site A glycoprotein (gp80) of Dictyostelium discoideum

Developmentally regulated cohesion of Dictyostelium discoideum can be blocked by the Fab fragment of antiserum prepared against a glycoprotein of about 80,000 daltons, gp80, purified from the membranes of developing amoebae. Immunoprecipitation of gp80 with this serum, from ³²P-labeled cell extracts of aggregating D. discoideum amoebae showed it to be a phosphoprotein. Serine phosphate was found in the molecule. All multiple isoelectric forms of gp80 were phosphorylated. Synthesis of the phosphorylated form of gp80 was found to be limited to the period of aggregation and coincided with the period of incorporation of [³⁵S]methionine into gp80. Phosphorylation could be rapidly inhibited by cycloheximide suggesting that phosphorylation occurs only on newly made gp80. No unphosphorylated gp80 could be detected in cell extracts.