Antibiotic resistance.
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Anti-Bacterial Agents↗
Biomedical subjects
H L Cooper
Publications and source records attributed to H L Cooper.
Complementary DNA cloning of a novel epithelial cell marker protein, HME1, that may be down-regulated in neoplastic mammary cells.
A full-length complementary DNA clone from a normal human mammary epithelial cell (strain 184) encoding a 25-kilodalton protein, HME1, was isolated. Expression of HME1 RNA appears to be limited to epithelial cells. The HME1 sequence has extensive sequence homology with bovine 14-3-3 protein, which is an activator of tyrosine and tryptophan hydroxylase. However, the tissue distribution, arrangement of charged amino acids, and location of potential phosphorylation sites of HME1 differ from those of 14-3-3. Compared with normal mammary epithelial cells, expression of HME1 RNA was dramatically low in two cell lines derived from human mammary carcinoma that were examined, and in a line of normal mammary epithelial cells transformed by oncogenes. HME1 therefore appears to be a cellular differentiation marker that may be down-regulated during neoplastic transformation.
14-3-3 Proteins↗
A cDNA encoding a muscle-type tropomyosin cloned from a human epithelial cell line: identity with human fibroblast tropomyosin TM1.
Tropomyosins (TM) expressed by human epithelial cells have only recently been characterized, and no sequence data for them has appeared. We cloned a cDNA encoding a high molecular weight, muscle-type TM from a LS174T human colon carcinoma epithelial cell cDNA library. On sequence analysis this cDNA (TMe1) was virtually identical to the previously reported sequence for human fibroblast TM1 encoded by the hTM beta gene. Expression of TM1/TMe1 mRNA and protein are low in epithelial cells compared with fibroblasts. The results indicate that cells of different developmental lineages (entodermal and mesodermal) can produce identical TM beta gene splice products while regulating expression of those transcripts in a lineage-specific way.
Base Sequence↗
T cell receptor activation induces rapid phosphorylation of prosolin, which mediates down-regulation of DNA synthesis in proliferating peripheral lymphocytes.
Prosolin is a major cytosolic phosphoprotein expressed prominently in rapidly proliferating human peripheral lymphocytes but produced at very low levels in resting (G0) PBL. It undergoes rapid phosphorylation upon treatment of growing cells with tumor-producing phorbol esters (TPA) and this phosphorylation event is correlated with a rapid down-regulation of DNA synthesis. In the present report we have studied various agents that, like TPA, act as partial or complete mitogens for G0 PBL and have determined their effect on phosphorylation of prosolin and on DNA synthesis in rapidly proliferating (IL-2-dependent) human PBL. Agents that activate the TCR (OKT3 and PHA), as well as agents that by-pass the receptor but activate biochemical pathways associated with TCR activation (TPA and Ca2(+)-ionophore), all produced rapid phosphorylation of prosolin and prompt down-regulation of DNA synthesis. Four phosphorylated forms of prosolin were produced, indicating activation of a complex phosphorylation pathway. Down-regulation of DNA synthesis did not lead to cell death or to permanent arrest, but was reversed after 24 to 48 h, and was not associated with any reduction in overall protein synthesis. Agents that bind to determinants closely connected to the TCR but without activating it (OKT4 and OKT8) had no effect on either prosolin phosphorylation or DNA synthesis. The results indicate that prosolin is an early target of the protein kinase activities induced by activation of the TCR in proliferating PBL, and suggest that its phosphorylation mediates the TCR signal, transmitting it into a biochemical pathway leading specifically to down-regulation of DNA synthesis. In G0 PBL, in which the negligible expression of prosolin precludes significant production of phosphorylated species, this inhibitory pathway is effectively blocked.
Antibodies, Monoclonal↗
A specific defect of prosolin phosphorylation in T cell leukemic lymphoblasts is associated with impaired down-regulation of DNA synthesis.
Prosolin is a major cytosolic phosphoprotein of proliferating normal PBL. Treatment of growing PBL with phorbol ester (12-O-tetradecanoylphorbol-13-acetate (TPA)) or calcium ionophore (A23187) for 1 h caused phosphorylation of prosolin with the production of up to four prominent phosphorylated forms differing in degree of phosphorylation and/or two-dimensional electrophoretic mobility (peptides B to E). Formation of these phosphopeptides coincided with rapid down-regulation of DNA synthesis. A23187 was particularly effective in inducing phosphorylation of the more highly phosphorylated peptides D and E, suggesting the existence of a (Ca2+)-activated mechanism in their phosphorylation. The T cell leukemia cell lines Jurkat, HuT-78, CCRF-CEM, and Molt-4 showed reduced to absent ability to phosphorylate prosolin peptides rapidly in response to A23187 and also showed diminished down-regulation of DNA synthesis. In leukemic cells treated with both TPA and A23187, peptides B and C were rapidly phosphorylated, but the phosphorylation of peptides D and E seen in normal PBL remained deficient. The T cell leukemic cells appear to have intact a TPA-activated mechanism for phosphorylating prosolin peptides B and C, but share an impairment of a specific Ca2(+)-activated mechanism, possibly a Ca2(+)-dependent protein kinase, required for phosphorylation of prosolin phosphopeptides D and E. The degree of rapid down-regulation of DNA synthesis was correlated with degree of phosphorylation of peptide E in PBL and in three of four T cell leukemic cell lines. Thus, rapid phosphorylation of prosolin may mediate responses to TPA and A23187 in normal proliferating PBL, including down-regulation of DNA synthesis. A deficiency of this pathway in leukemic T cells may impede their response to physiologic growth regulatory signals utilizing this pathway and contribute to unrestrained cell growth.
Calcimycin↗
Tropomyosins of human mammary epithelial cells: consistent defects of expression in mammary carcinoma cell lines.
Suppression of synthesis of specific tropomyosin (TM) isoforms occurs commonly in human, murine, and avian fibroblasts transformed by retroviral oncogenes or other modalities. The resulting deficiency or altered distribution of TMs may predispose the cells to microfilament instability and contribute to expression of the transformed phenotype. In this study we have asked whether defects in TM expression had relevance to human neoplasia, which arises most often in cells of the epithelial lineage rather than in fibroblasts and often is unrelated to demonstrable expression of oncogenes. TMs were characterized in normal primary human mammary epithelial cells (HMEC) and in an immortalized nontumorigenic cell line derived from them. Seven TM isoforms were identified in primary HMEC, two of which may be unique to epithelial cells. Immortalized nontumorigenic HMEC expressed the same array of isoforms. Of six established human breast carcinoma cell lines studied, all failed to express the Mr 39,000 TM isoform and five of six also lacked expression of either the Mr 38,000 or 35,000 isoform. Northern blot analysis with probes specific for the 1.1-kilobase mRNA of fibroblast TM1 detected a mRNA of this size in normal HMEC. This mRNA, which probably encodes the Mr 39,000 TM missing from all the carcinoma lines, was absent from five of the six breast cancer cell lines. These results indicate that abnormalities in TM expression in neoplastic cells are not limited to fibroblasts. The high frequency and consistent nature of such abnormalities among cell lines derived from human breast cancer raises the possibility that such abnormalities in expression of a major cytoskeletal protein may play a role in human neoplasia.
Actins↗
Human peripheral lymphocyte growth regulation and response to phorbol esters is linked to synthesis and phosphorylation of the cytosolic protein, prosolin.
Prosolin is a major cytosolic protein (Mr 18400, isoelectric point 5.9) first reported in HL-60 promyelocytic leukemia cells. It is rapidly phosphorylated (15 to 30 min) in response to TPA treatment as an early event in a sequence that leads to cessation of cell proliferation and to differentiation of promyelocytes into monocytes. In our study we examined the expression of prosolin in human peripheral lymphocytes and investigated the effects of TPA treatment on prosolin phosphorylation and on lymphocyte proliferation. Prosolin was not expressed in resting PBL but was induced after 24 to 36 h of PHA stimulation, simultaneously with induction of DNA synthesis. In rapidly proliferating (IL-2 dependent) PBL prosolin was a major cytosolic component, comprising 0.5% of total cytosolic protein, of which approximately 28% was phosphorylated. Expression of prosolin decreased again when either mitogen-induced or IL-2-dependent proliferation diminished during extended periods in culture. Thus, expression of prosolin is correlated with periods when PBL are cycling through S-phase. TPA treatment of IL-2-dependent PBL at the peak of their growth caused phosphorylation of about two-thirds of preexisting unphosphorylated prosolin within 1 h. This was accompanied by cessation of cell proliferation, as indicated by measurements of TdR incorporation. Although TPA has well known mitogenic effects in lymphocytes during initial activation, this result shows that it exerts an antiproliferative effect in rapidly dividing PBL. It is suggested that increased phosphorylation of prosolin may be an initiating event in the antiproliferative response to TPA, which would occur only in proliferating lymphocytes expressing prosolin.
Cell Differentiation↗
Purification and composition of the human tumor-associated glycoprotein (TAG-72) defined by monoclonal antibodies CC49 and B72.3.
Human mammary and other carcinoma cells secrete and express on their cell surfaces complex, mucin-like glycoproteins (Mr greater than 10(6] that are recognized as tumor-associated antigens by a variety of monoclonal antibodies (MAbs). One such MAb, B72.3, has been extensively studied as to range of reactivity for a variety of carcinomas versus normal tissues. The B72.3-reactive antigen, designated tumor-associated glycoprotein (TAG)-72, which is a high-molecular-weight mucin, was partially purified and used as immunogen to produce second generation anti-TAG-72 MAbs. One of these second generation MAbs, CC49, was chosen to be used to develop a procedure to yield preparative amounts of purified antigen suitable for analyzing amino acid sequence. One of the reasons for the selection of CC49 for these studies is that it demonstrated a higher Ka for TAG-72 compared with B72.3. Xenografts of LS174T cells (a human colon carcinoma cell line) grown in nude mice were solubilized, extracted with several chaotropic agents and treated with perchloric acid. The acid soluble antigen was subjected to MAb CC49 affinity chromatography, gel filtration, and ion-exchange chromatography using fast protein liquid chromatography and high-performance liquid chromatography methodologies. A double-determinant liquid competition radioimmunoassay for TAG-72 showed greater than a 1000-fold purification. Radiolabeled protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated an apparently homogeneous high molecular weight mucin and a small amount of an additional protein with an apparent Mr of 63,000. Chemical deglycosylation using trifluoromethanesulfonic acid yielded low molecular weight proteins, which could be analyzed for amino acid sequence, and also became susceptible to tryptic digestion. The amino acid composition of the purified TAG-72 mucin was similar to that of other purified mucins.
Amino Acids↗
Disordered metabolism of microfilament proteins, tropomyosin and actin, in mouse mammary epithelial cells expressing the Ha-ras oncogene.
Synthesis of 37- and 41-kD tropomyosins (TM) is suppressed in fibroblasts expressing transforming oncogenes, which may contribute to the deranged microfilament structure seen in such cells (Cooper et al., Mol. Cell. Biol. 5, 972, 1985). To determine whether TM metabolism was also deranged when epithelial cells expressed a transforming oncogene, we studied cultured mouse mammary epithelial cells which express the activated c-Ha-ras oncogene either constitutively or through glucocorticoid activation of the oncogene linked to a mouse mammary tumor virus (MMTV) promoter. The epithelial cells expressed three major TM species, one at 37 kD and two at 35 kD, but lacked expression of a 41 kD TM typically found in fibroblasts. In oncogene-expressing cells synthesis of the 37 kD TM was not suppressed but synthesis of actin was increased 2-fold. Actin entered the cytoskeleton (CSK) normally, but TM accumulated in the CSK with a 50% reduction from normal in TM:actin ratio. Thus, in both epithelial cells and fibroblasts expressing transforming ras oncogenes, the metabolism of TM and actin is disordered so as to produce TM-deficient cytoskeletal structures which may be functionally or structurally defective. However, the result is achieved by different effects on metabolism of these proteins in the two cell types, suggesting that cellular responses to oncogene actions may be conditioned by the state of cell differentiation.
Actins↗
Suppression of synthesis and utilization of tropomyosin in mouse and rat fibroblasts by transforming growth factor alpha: a pathway in oncogene action.
Two events which commonly occur during transformation of murine and avian fibroblasts by retroviral oncogenes are production of transforming growth factor alpha (TGF-alpha) and suppression of tropomyosin synthesis. TGF has been proposed as a mediator of transformation through autocrine stimulation. Suppression of tropomyosin synthesis may contribute to the transformed phenotype through destabilization of actin microfilaments and cytoskeletal derangement. To determine whether suppression of tropomyosin synthesis might be a consequence of the action of TGF-alpha we studied tropomyosin synthesis in rat (normal rat kidney) and mouse (NIH3T3) fibroblasts treated with TGF-alpha. In a serum-containing system, addition of TGF-alpha or epidermal growth factor to normal rat kidney monolayers in subnanomolar concentrations induced morphological changes consistent with transformation. These changes were accompanied by prominent suppression of synthesis of Mr 36,000 and 39,000 tropomyosins. Similar suppression was observed in NIH3T3 cells. Inhibition of tropomyosin synthesis began almost immediately after addition of TGF-alpha and became progressively more pronounced during the succeeding 48 h. Suppression of tropomyosin synthesis was correlated with reduced expression of 1.1- and 1.8-kilobase tropomyosin mRNAs in both TGF-treated normal rat kidney cells and v-Ki-ras-transformed NIH3T3 cells. Rapid onset of a specific block in utilization of newly synthesized tropomyosin for formation of cytoskeletal elements was also demonstrated following TGF-alpha treatment. The evidence suggests that this block may be a specific effect of TGF-alpha treatment and that reduced expression of tropomyosin gene products may be either an independent event or a regulatory consequence of the block to utilization. The data support the conclusion that suppression of tropomyosin synthesis in cells transformed by a number of retroviral oncogenes results from the autocrine action of TGF-alpha.
Animals↗
Proteins associated with B lymphocyte hyperactivity in New Zealand black mice.
New Zealand Black (NZB) mice exhibit polyclonal B cell activation and elevated immunoglobulin production, an abnormality associated with the spontaneous autoimmune disease that affects this strain. To further our understanding of this abnormality of B cell differentiation and maturation, we have employed two-dimensional polyacrylamide gel electrophoresis to analyze the proteins synthesized by lymphocytes of several strains. Two proteins were produced by lymphocytes from NZB mice but not those from normal strains. One was a 16 kd protein with a pI of 5.1, and the other was a 27.5 kd protein with a pI of 4.5. The presence of the xid gene on the NZB background suppressed production of both proteins. They were synthesized by spleen cells but not by bone marrow or lymph node cells, and production was restricted to enlarged B lymphocytes. p16 was synthesized by normal mouse strain B cells upon stimulation with LPS. The 27.5 kd protein was shown to be secreted. On the basis of partial amino acid sequence determination of proteins eluted from gels, p27.5 was identified as J chain and p16 as the C terminal fragment of mu-chain. The synthesis of two other proteins, 13 kd and 18 kd in size, was elevated in NZB spleen lymphocytes. The 18 kd protein was identified as translation initiation factor eIf-4D. The increased level of this protein may be related to the upregulation of immunoglobulin synthesis.
Amino Acid Sequence↗
Identification and characterization of the nonphosphorylated precursor of pp17, a phosphoprotein associated with phorbol ester induction of growth arrest and monocytic differentiation in HL-60 promyelocytic leukemia cells.
Treatment of HL-60 promyelocytic leukemia cells with the tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), causes rapid phosphorylation and dephosphorylation of pp17, a 17-20-kDa, pI 5.5 cytosolic protein, as an early event in a response sequence leading to growth arrest and terminal differentiation into monocytes (Feuerstein, N., and Cooper, H. L., (1984) J. Biol. Chem. 259, 2782-2788). In the present study, we have identified the nonphosphorylated precursor to pp17 by tryptic peptide mapping of single proteins recovered from two-dimensional gels. The pI of the precursor, p17, was 5.9, and the apparent Mr of both p17 and pp17 was 18,400 by SDS-polyacrylamide gel electrophoresis (one dimension). p17 was shown to be a major cytosolic protein, comprising about 0.5% of steady state labeled protein in that fraction. Both p17 and pp17 were found exclusively in the cytosol (detergent-released SOL) and were not detected in membranes, cytoskeleton, or nuclei. In untreated cells, about 90% of the protein was present in the nonphosphorylated form. Upon TPA treatment, pre-existing p17 was rapidly phosphorylated to pp17. After 15 min, the two forms were nearly equal in quantity. This corresponds to phosphorylation, within that period, of about 0.2% of total cytosolic protein, represented by a single species. The maximum level of pp17 was reached within 1 h, with pp17 exceeding p17 by about 25%. Quantitatively, therefore, the phosphorylation of p17 to pp17 is one of the most prominent early biochemical responses to TPA treatment. Available data indicate that p17 predominates in rapidly proliferating cells, while phosphorylation to pp17 occurs where cell growth is modified by TPA or other agents. Thus, the p17/pp17 system is potentially a major mechanism for intracellular propagation of growth regulatory signals. We propose the name, prosolin, for this prominent cytosolic protein.
Carrier Proteins↗
Analysis of ras oncogene products by two-dimensional gel electrophoresis: evidence for protein families with distinctive molecular forms.
Protein products of the ras family of oncogenes were immunoprecipitated by an anti-p21 monoclonal antibody, separated by two-dimensional gel electrophoresis and subsequently detected by western immunoblot analysis using the same anti-p21 monoclonal antibody as a probe. Using this method, a 21 kDa oncogene protein (p21) was detected and characterized in cell lines containing Harvey (Ha), Kirsten (Ki), neuroblastoma (N), or cellular (proto) ras genes. The ras gene products from all cell types occurred with multiple forms differing in size, charge or in both parameters. Transforming ras oncogene proteins occurred in easily identifiable groups that were different from each other in molecular weight and charge, were distinctive for each ras gene type and were different from cellular ras equivalents. Similar, but not identical, family groups occurred in different cell types containing the same oncogene. The reproducible occurrence of unpredicted p21 forms suggests that previously unreported post-translational processing steps may be associated with the synthesis of certain oncogene products. This immunoprecipitation/two-dimensional gel/western blot technique is a simple method for the identification and characterization of p21 gene products.
Amino Acid Sequence↗
Effect of epidermal growth factor and 12-O-tetradecanoylphorbol-13-acetate on the phosphorylation of soluble acidic proteins in A431 epidermoid carcinoma cells.
Treatment of [32P]phosphate prelabeled intact human A431 epidermoid carcinoma cells with epidermal growth factor (EGF, 100 ng/ml) or 12-O-tetradecanoylphorbol-13-acetate (TPA, 10(-7)M) resulted in a selective enhancement in the phosphorylation of the following soluble acidic proteins: a phosphoprotein with a molecular weight of 17,000 (pp17; similar notation used throughout) pI approximately 5.5); pp27 (pI approximately 5.5); pp34 (pI approximately 6.2); and pp80 (pI approximately 4.5) as detected by two-dimensional gel electrophoresis. EGF or TPA induced a 4- to 6-fold increase in the phosphorylation of pp17 and a 2- to 4-fold increase in the phosphorylation of pp27, pp34, and pp80 within 15 min after treatment of subconfluent A431 cells. Alkali treatment of the gels removed most of the incorporated [32P] phosphate from the phosphoproteins, including pp27, pp34, and pp80; however, the phosphoester bond in pp17 was stable to alkaline hydrolysis since there was no removal of [32P]phosphate from this protein. Treatment of A431 cells with dibutyryl cyclic adenosine 3':5'-monophosphate (1 mM) also increased the phosphorylation of pp17, pp27, and pp34 but not of pp80. Activation of endogenous calcium- and phospholipid-dependent protein kinase C in the cytosol of A431 cells in a cell-free system resulted in the enhanced phosphorylation of pp27, pp34, and pp80 but not of pp17 while exogenous addition of the catalytic subunit of cyclic adenosine 3':5'-monophosphate-dependent protein kinase to cytosol preparations resulted in the phosphorylation of pp17, pp27, and pp34, but not of pp80. These results demonstrate that at least four soluble acidic proteins are phosphorylated in A431 cells in response to either EGF or TPA in vivo suggesting that these two agents may exert part of their biological effects on A431 cells through a biochemical pathway involving the phosphorylation of several common proteins; moreover, the studies suggest that these four acidic proteins may be substrates in vitro for protein kinase C and/or a cyclic adenosine 3':5'-monophosphate-dependent protein kinase.
Bucladesine↗
Lymphocyte blastogenesis. Post-transcriptional controls of protein synthesis.
To better understand the regulation of macromolecular synthesis in the response of lymphocytes to a mitogen, we have used two-dimensional electrophoresis to search for specificity in the early increase seen in protein synthesis in human lymphocytes treated with phytohemagglutinin and examined the role of new RNA synthesis in this response. Our results confirm a major increase in overall protein synthesis after 4 h of phytohemagglutinin treatment. A further disproportionate increase in the synthetic rates of certain polypeptides was observed using two-dimensional polyacrylamide gel electrophoresis. While actinomycin-D reduced protein synthesis to a level below that of untreated cells, phytohemagglutinin nonetheless enhanced total protein synthesis even in the presence of actinomycin. Some, but not all, of the disproportionate increases in synthesis seen for certain polypeptides are blocked by actinomycin. These results imply the existence of multiple mechanisms controlling protein synthesis early in the course of lymphocyte stimulation. At least some of these do not require new RNA synthesis and thus operate at a post-transcriptional level.
Blood Proteins↗
Phorbol ester effect in platelets, lymphocytes, and leukemic cells (HL-60) is associated with enhanced phosphorylation of class I HLA antigens. Coprecipitation of myosin light chain.
Phorbol-12-myristate-13-acetate (PMA) is a potent tumor promoter. However, the mechanism of its effect is still unknown. In the present study we use two dimensional gel electrophoresis to show that the PMA effect on platelets is associated with enhanced phosphorylation of a series of polypeptides at 44K which migrate close to but distinct from a previously reported 47K protein. We identified these proteins as the class I molecules of the human histocompatibility antigens (HLA A,B). We further demonstrate that the PMA effect is also associated with a dramatic phosphorylation of HLA antigens in HL-60 leukemic cells and in human lymphocytes, showing that an increase in phosphorylation of HLA antigens is intimately related to the signal of PMA in various cellular systems. Immunoprecipitation of HLA proteins resulted in coprecipitation of phosphorylated myosin light chain (20K). HLA antigens are transmembrane proteins which interact with cytoskeletal elements, probably via their intracellular region, which has been previously shown to be phosphorylated. It is suggested that phosphorylation of HLA membrane proteins may represent an important mechanism in the effects induced by PMA.
Blood Platelets↗
Suppression of tropomyosin synthesis, a common biochemical feature of oncogenesis by structurally diverse retroviral oncogenes.
To identify proteins whose production may be altered as a common event in the expression of structurally diverse oncogenes, we compared two-dimensional electropherograms of newly synthesized proteins from NIH/3T3 cell lines transformed by a variety of retroviral oncogenes, from cellular revertant lines, and from a line (433.3) which expresses the v-ras oncogene in response to corticosteroids. Most alterations in the synthesis of specific proteins detected by this approach appeared to be the result of selection during prolonged cultivation and were probably unrelated to the transformation process. However, we detected seven proteins whose synthesis was strongly suppressed in cell lines transformed by each of the six retroviral oncogenes we studied and whose production was fully or partially restored in two cellular revertant lines. Suppression of two of these proteins was also correlated with the initial appearance of morphological alteration during corticosteroid-induced oncogene expression in 433.3 cells. These proteins (p37/4.78 and p41/4.75) were identified as tropomyosins, a group of at least five cytoskeletal proteins. Transformation by the papovaviruses simian virus 40 and polyomavirus caused no suppression of synthesis of these tropomyosins. This indicates that suppression of tropomyosin synthesis is not a nonspecific response by cells to being forced to grow with the transformed phenotype but is specifically associated with oncogenesis by diverse retroviral oncogenes. The results are consistent with the hypothesis that the different biochemical processes initiated by expression of structurally diverse retroviral oncogenes may converge on a limited number of common targets, one of which is the mechanism which regulates the synthesis of tropomyosins.
Animals↗
Cell-free system studies on the phosphorylation of the 17,000-20,000 dalton protein induced by phorbol ester in human leukemic cells and evidence for a similar event in virally transformed murine fibroblasts.
We have shown previously that a prominent early signal in the phorbol-12-myristate-13-acetate (PMA) effect on leukemic cells as well as on other malignant cells is a rapid and dramatic increase in the turnover of phosphate in a Mr 17,000 to 20,000 cytosolic protein and a moderate increase in turnover of phosphate in a Mr 27,000 protein, as detected in the intact cells by 2-dimensional gel electrophoresis. To further elucidate the mechanism of this phosphorylation event, we have examined the protein kinases which can reconstitute this event in a cell-free system. Activation of the endogenous Ca2+-activated phospholipid-dependent protein kinase (Ca-PL-PK) as well as addition of purified Ca-PL-PK to the cytosol of HL-60 leukemic cells resulted in enhanced phosphorylation of phosphoprotein Mr 27,000 (PP27) but did not affect the phosphorylation of phosphoprotein Mr 17,000 to 20,000 (PP17-20). In contrast, PP17-20 was heavily phosphorylated under cell-free conditions by the catalytic subunit of cAMP-dependent protein kinase (cAMP-PK). Exposure of intact cells to dibutyryl-cAMP resulted in increased phosphorylation of PP17-20. These conditions also enhanced the phosphorylation of PP27, showing that PP27 can act as a substrate for both Ca-PL-PK and cAMP-PK under cell-free conditions. Tryptic digest analysis of PP17-20 showed that one of four phosphopeptides is preferentially phosphorylated in PMA-induced PP17-20. An additional phosphopeptide was phosphorylated in cAMP-PK-catalyzed PP17-20. Thus, cAMP-PK alone mimics the effect of PMA on phosphorylation of PP17-20, but it introduces additional modifications. The precise role of this kinase in PMA-induced phosphorylation of PP17-20 remains to be clarified. We found further that enhanced phosphorylation of PP17-20 is also associated with malignant transformation of NIH/3T3 cells transformed by V-rasKi oncogene of Kirsten sarcoma virus. The tryptic phosphopeptide map of PP17-20 (phosphorylated in vivo) in the transformed cells was similar to that of PP17-20 in PMA-treated HL-60 cells but not to that induced by cAMP-PK, suggesting that the process activated by PMA which leads to phosphorylation of PP17-20 resembles an intrinsic cellular process which is enhanced in certain malignant cells.
Animals↗