Phosphorylation of specific polypeptides in isolated murine splenocyte nuclei which is controlled by cyclic nucleotides

Murine splenocyte nuclei were phosphorylated with a less than 10(-5) M concentration of [gamma-32P]ATP at 0 degrees C and the phosphorylated nuclear proteins were analyzed by SDS-polyacrylamide gel slab electrophoresis and Sephadex gel filtration column chromatography. Two polypeptides of 10K and 11K daltons were predominantly phosphorylated. These polypeptides were likely linked by a disulfide bond to form a nonhistone protein of 21K daltons. Both phosphoserine and phosphothreonine were detected in the hydrolysate of the 10.5K dalton polypeptide, while phosphoserine was predominant in the 10K dalton polypeptide. Maximal activation of phosphorylation by cAMP of both polypeptides was shown at a concentration of 10(-6) M. On the contrary, cGMP activated phosphorylation of the 10K dalton polypeptide at 10(-8) M and at 10(-4) M. The phosphorylation of the 10.5K polypeptide was not activated by 10(-4) M cGMP and suppression of the phosphorylation was seen in both polypeptide chains by cAMP at higher concentrations.     

Reversal of thrombin-induced myosin phosphorylation and the assembly of cytoskeletal structures in platelets by the adenylate cyclase stimulants prostaglandin D2 and forskolin

Stimulation of platelets by thrombin causes an increase in the amount of cytoskeleton proteins insoluble in 1% Triton X-100, i.e. myosin, actin, actin-binding protein, an alpha-actinin-like protein of Mr = 105,000, unidentified polypeptides of Mr = 150,000, 31,00, and under some conditions, 56,000. Concurrently the Mr = 20,000 light chains of myosin and a cytoplasmic Mr = 42,000 polypeptide are phosphorylated, presumably by calmodulin-Ca2+-dependent myosin light chain kinase and a phospholipid-Ca2+-dependent kinase, respectively. The adenylate cyclase stimulators prostaglandin D2 (PGD2) and forskolin increased platelet cyclic AMP and prevented the phosphorylation of these polypeptides and the increase in Triton-insoluble cytoskeleton proteins. When added to platelets after stimulation by thrombin they caused rapid complete reversal of myosin light chain and Mr = 42,000 polypeptide phosphorylation; simultaneously the association of myosin with the cytoskeleton proteins and the increase in the content of each of the Triton-insoluble cytoskeleton proteins (except the Mr = 56,000 polypeptide) was reversed. The amount of Triton-insoluble myosin was affected more readily by PGD2 or forskolin than were the other proteins. Increasing thrombin from 0.1 to 1.0 unit/ml inhibited all the responses to PGD2 and forskolin possibly due to concentration-dependent effects of thrombin that inhibit adenylate cyclase. These results suggest that cytoskeleton assembly and activation of the contractile apparatus in intact platelets are readily reversible by cyclic AMP-dependent reactions.     

Purification and characterization of a 400-kDa nonhistone chromatin protein that serves as an effective phosphate acceptor for casein kinase II from Ehrlich ascites tumor cells

A nonhistone chromatin protein (NHCP) has been purified to homogeneity from a 0.5 M NaCl extract of Ehrlich ascites tumor cell (EAT cell) nuclei as a phosphate acceptor for casein kinase II using ion-exchange column chromatographies and Sephacryl S300 gel filtration. The purified NHCP (approximate Mr = 400,000) was found to be a tetramer of an Mr = 98,000 polypeptide (pI = 6.9) and to have high contents of glycine (15%) and serine (11.6%). This protein (designated as 400-kDa NHCP) was highly phosphorylated by casein kinase II (Mr = 130,000), but not by histone kinase. Casein kinase II phosphorylated only seryl residues of the purified 400-kDa NHCP. The NHCP bound with DNA, but not with RNAs, and the DNA binding ability of the protein was reduced when it was phosphorylated by casein kinase II. Moreover, we found that (a) the 400-kDa NHCP is present in large quantities in malignant mouse cells, such as EAT, EL-4, and Meth-A cells, but only slightly in normal tissues and cells; (b) the protein level is rapidly increased when mouse lymphocytes are treated with recombinant interleukin 2 (T cell growth factor) or concanavalin A; and (c) the kinase responsible for the 400-kDa NHCP phosphorylation in the chromatin of various mouse cells is a casein kinase II. These experimental results suggest that the 400-kDa NHCP acts as an effective phosphate acceptor for casein kinase II at the chromatin level and that an increased phosphorylation of the protein by the kinase may be implicated in the progress of cell differentiation and proliferation.     

Rapid phosphorylation of the L-myc protein induced by phorbol ester tumor promoters and serum.

We have examined post-translational modification of the L-myc protein using polyclonal and monoclonal antibodies against a peptide well conserved in the predicted amino acid sequences of the c-myc, N-myc and L-myc genes. These antibodies precipitate three polypeptides of Mr 60-66,000 from [35S]methionine or [32P]orthophosphate-labelled human small cell lung cancer cell lines expressing amplified L-myc genes, but not the other myc genes. Treatment of the L-myc immunoprecipitates with alkaline phosphatase prior to electrophoresis converts the three methionine-labelled polypeptides into a single band migrating at Mr 59,000, and efficiently removes radioactivity from the 32P-labelled L-myc protein, suggesting that, in contrast to the c-myc and N-myc proteins, the L-myc polypeptide heterogeneity is due to differential phosphorylation of a common precursor. When the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or serum is added to cultures of U-1690 cells the Mr 66,000 polypeptide is rapidly enriched while the Mr 60,000 form is decreased in the L-myc immunoprecipitates. This effect is correlated with the ability of phorbol ester and diacylglycerol analogues to activate protein kinase C. The TPA-induced phosphorylation of the L-myc protein occurs in a protein synthesis-independent manner as it is not inhibited by cycloheximide or anisomycin. These data indicate that the phosphorylation of the L-myc nuclear oncoprotein is modulated in response to TPA via a rapid signal transduction system involving protein kinase C. This mechanism could play an important role in the response of lung cells to e.g. bombesin-related growth factors.     

Protein phosphorylation in Escherichia coli and purification of a protein kinase

More than 40 protein species including RNA polymerase were found to be phosphorylated in Escherichia coli on analyses of 32P-labeled cell lysates by single and two-dimensional gel electrophoresis and autoradiography. The protein species and the level of phosphorylation varied depending on the cell growth phase. With [gamma-32P]ATP as a substrate, cell lysates phosphorylated endogenous proteins in vitro which were predominantly phosphorylated in vivo. Both serine and threonine were the major phosphate acceptors in whole cell lysates. Starting from a partially purified RNA polymerase preparation with the protein phosphorylation activity and using an E. coli protein with an apparent Mr = 90K (K represents X 1000) as the substrate, we purified a protein kinase with a native Mr approximately 120K to apparent homogeneity. The protein kinase is either a heterodimer of 61K and 66K polypeptides or a homodimer of one of these polypeptides. We also isolated a 100K protein with self-phosphorylation activity.     

ANALYSIS OF POLYPEPTIDES ASSOCIATED WITH SHOOT FORMATION IN TOBACCO CALLUS CULTURES

Callus lines of Nicotiana tabacum were selected for competence and lack of competence in shoot formation. Changes in total and chromosomal polypeptides in these shoot-forming and nonshoot-forming tobacco cultures were examined by twodimensional polyacrylamide gel electrophoresis. Qualitative and quantitative differences in total, nonhistone chromosomal, and basic chromosomal polypeptides were evident throughout the 7-d test period. The analysis of total proteins identified polypeptides specific to shoot-forming and nonshoot-forming tissue during the 7-d sampling period. A small number of basic chromosomal proteins were found solely in shoot-forming or nonshoot-forming tissue. One basic chromosomal protein was detected in only nonshoot-forming tissue at all sampling times. Two proteins, although present in shoot-forming tissue, were present at elevated levels in the nonshoot-forming cultures. No temporal changes in basic proteins over the 7-d incubation period were observed. Qualitative differences in total nonhistone chromosomal polypeptides in the shoot-forming and nonshoot-forming tissue were also observed. Differences in chromosomal polypeptides were observed. In contrast to the basic chromosomal proteins, temporal variation in the nonhistone chromosomal polypeptides was demonstrated. Throughout the 7-d sampling period, 29 and 12 nonhistone chromosomal polypeptides varied qualitatively in shoot-forming and nonshoot-forming callus cultures, respectively. In vitro labeling with ³²P-orthophosphate indicated that approximately 1.0% and 0.3% of the nonhistone chromosomal proteins were phosphorylated in the shoot-forming and nonshoot-forming cultures. Of these phosphorylated polypeptides, one was present in nonshoot-forming tissue and three were detected only in the shoot-forming tissue. Phosphorylation occurred at serine or threonine residues.     

Autophosphorylation of Calmodulin-Kinase II in Synaptic Junctions Modulates Endogenous Kinase Activity

Previous studies have purified from brain a Ca2+/calmodulin-dependent protein kinase II (designated CaM-kinase II) that phosphorylates synapsin I, a synaptic vesicle-associated phosphoprotein. CaM-kinase II is composed of a major Mr 50K polypeptide and a minor Mr 60K polypeptide; both bind calmodulin and are phosphorylated in a Ca2+/calmodulin-dependent manner. Recent studies have demonstrated that the 50K component of CaM-kinase II and the major postsynaptic density protein (mPSDp) in brain synaptic junctions (SJs) are virtually identical and that the CaM-kinase II and SJ 60K polypeptides are highly related. In the present study the photoaffinity analog [alpha-32P]8-azido-ATP was used to demonstrate that the 60K and 50K polypeptides of SJ-associated CaM-kinase II each bind ATP in the presence of Ca2+ plus calmodulin. This result is consistent with the observation that these proteins are phosphorylated in a Ca2+/calmodulin-dependent manner. Experiments using 32P-labeled peptides obtained by limited proteolysis of 60K and 50K polypeptides from SJs demonstrated that within each kinase polypeptide the same peptide regions contain both autophosphorylation and 125I-calmodulin binding sites. These results suggested that the autophosphorylation of CaM-kinase II could regulate its capacity to bind calmodulin and, thus, its capacity to phosphorylate substrate proteins. By using 125I-calmodulin overlay techniques and sodium dodecyl sulfate-polyacrylamide gel electrophoresis we found that phosphorylated 50K and 60K CaM-kinase II polypeptides bound more calmodulin (50-70%) than did unphosphorylated kinase polypeptides. Levels of in vitro CaM-kinase II activity in SJs were measured by phosphorylation of exogenous synapsin I. SJs containing highly phosphorylated CaM-kinase II displayed greater activity in phosphorylating synapsin I (300% at 15 nM calmodulin) relative to control SJs that contained unphosphorylated CaM-kinase II. The CaM-kinase II activity in phosphorylated SJs was indistinguishable from control SJs at saturating calmodulin concentrations (300-1,000 nM). These findings show that the degree of autophosphorylation of CaM-kinase II in brain SJs modulates its in vitro activity at low and possibly physiological calmodulin concentrations; such a process may represent a mechanism of regulating this kinase's activity at CNS synapses in situ.     

DNA-stimulated protein phosphorylation in HeLa whole cell and nuclear extracts

Incorporation of 32P from gamma-labeled ATP into a number of polypeptides in HeLa whole cell and nuclear extracts was dependent on added double-stranded DNA or poly(dI-dC), but not denatured or supercoiled DNA. DNA-dependent phosphorylation of a high Mr endogenous substrate could be reconstituted from the precipitate formed after incubation of whole cell extracts with DNA. Fractionation of extracts by phosphocellulose or DEAE-Sephacel chromatography yielded preparations that phosphorylated casein as well as endogenous polypeptides in a DNA-dependent manner. These results are consistent with the existence of a novel protein kinase in HeLa cells that is highly dependent upon the presence of double-stranded DNA for efficient phosphorylation of a variety of substrates.     

Phosphorylation of RNA polymerase I-associated polypeptides of Tetrahymena pyriformis

In the ciliate Tetrahymena pyriformis phosphorylation of RNA polymerase I [EC 2.7.7.6] and of polymerase-associated polypeptides was investigated in growing and growth-arrested cultures which differ widely in their rates of rRNA synthesis. Several putative subunits of RNA polymerase I (of 180, 21.5, and 19.5 kDa) and a polymerase-associated polypeptide of 27 kDa were found to be phosphorylated, independent of the growth conditions. However, an additional enzyme-associated polypeptide of 26 kDa was intensively labeled with 32P only after arrestment of growth by starvation. The molar quantities of both phosphorylated, enzyme-associated polypeptides thereby did not differ in growing and growth-arrested cultures, and the specific 32P-labeling of cellular ATP remained nearly unchanged under the different culture conditions. These findings indicate a selective, reversible phosphorylation of the RNA polymerase I-associated 26 kDa polypeptide correlated with conditions of repressed rRNA synthesis induced by the starvation procedure. In vitro phosphorylation in macronuclei isolated from growing and growth-arrested cultures using [gamma-32P]ATP revealed essentially the same pattern of labeling of the enzyme-associated polypeptides of 27 and 26 kDa as it was found in vivo.     

Polypeptide domains of ADP-ribosyltransferase obtained by digestion with plasmin

Proteolysis by plasmin inactivates bovine ADP-ribosyltransferase; therefore, enzymatic activity depends exclusively on the intact enzyme molecule. The transferase was hydrolyzed by plasmin to four major polypeptides, which were characterized by affinity chromatography and N-terminal sequencing. Based on the cDNA sequence for human ADP-ribosyltransferase enzyme [Uchida, K., Morita, T., Sato, T., Ogura, T., Yamashita, R., Noguchi, S., Suzuki, H., Nyunoya, H., Miwa, M., & Sugimura, T. (1987) Biochem. Biophys. Res. Commun. 148, 617-622], a polypeptide map of the bovine enzyme was constructed by superposing the experimentally determined N-terminal sequences of the isolated polypeptides on the human sequence deduced from its cDNA. Two polypeptides, the N-terminal peptide (Mr 29,000) and the polypeptide adjacent to it (Mr 36,000), exhibited binding affinities toward DNA, whereas the C-terminal peptide (Mr 56,000), which accounts for the rest of the transferase protein, bound to the benzamide-Sepharose affinity matrix, indicating that it contains the NAD+-binding site. The fourth polypeptide (Mr 42,000) represents the C-terminal end of the larger C-terminal fragment (Mr 56,000) and was formed by a single enzymatic cut by plasmin of the polypeptide of Mr 56,000. The polypeptide of Mr 42,000 still retained the NAD+-binding site. The plasmin-catalyzed cleavage of the polypeptide of Mr 56,000-42,000 was greatly accelerated by the specific ligand NAD+. Out of a total of 96 amino acid residues sequenced here, there were only 6 conservative replacements between human and bovine ADP-ribosyltransferase.