Jonathan A. Cooper

Epidermal growth factor induces rapid tyrosine phosphorylation of proteins in A431 human tumor cells

Addition of EGF to A431 cells at physiological concentrations causes a rapid three- to four-fold increase in the abundance of phosphotyrosine in cellular protein. The increase is essentially complete within 1 min and is maintained for several hours. No change in phosphotyrosine levels is found with fibroblast growth factor or insulin. Two phosphoproteins (molecular weights of 39 and 81 kd) containing phosphotyrosine appear de novo upon administration of EGF to A431 cells. The EGF receptor itself is a phosphoprotein containing phosphotyrosine as well as phosphoserine and phosphothreonine. Changes in the phosphorylation pattern of the EGF receptor are seen upon treatment of A431 cells with EGF. Increased phosphorylation of tyrosine is the most rapid response of cells to EGF known, and may play an important role in the biological effects of EGF.

Changes in protein phosphorylation in Rous sarcoma virus-transformed chicken embryo cells.

Rous sarcoma virus encodes a tyrosine-specific protein kinase (p60src) which is necessary for cell transformation. To identify substrates for this kinase, we set out to detect phosphotyrosine-containing proteins in Rous sarcoma virus-transformed chicken embryo cells, making use of the known alkali stability of phosphotyrosine. 32P-labeled phosphoproteins were separated by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gels were then incubated in alkali. Using this procedure with normal cells, we detected a total of about 190 alkali-resistant phosphoproteins. In Rous sarcoma virus-transformed cells, five phosphoproteins were found which were not detectable in normal cells. Two of these are probably structural proteins of the virus. The other three transformation-dependent phosphoproteins, and four other phosphoproteins which were elevated by transformation, all contained phosphotyrosine. Increased phosphorylation of these proteins did not occur with cells infected with a mutant Rous sarcoma virus, temperature sensitive for transformation, grown at the restrictive temperature. We conclude that these seven proteins are probably substrates of p60src, although they may be substrates for other tyrosine-specific protein kinases activated by p60src.

Similar effects of platelet-derived growth factor and epidermal growth factor on the phosphorylation of tyrosine in cellular proteins

Platelet-derived growth factor (PDGF) stimulates the phosphorylation of proteins at tyrosine when added to quiescent 3T3 cells, as evidenced by the increase in the amount of phosphotyrosine, relative to phosphoserine and phosphothreonine, in cellular proteins. The increase was detected within 1 min of adding PDGF and was maximal by 5 min. This effect showed the same dependence on PDGF concentration as did association of 125I-PDGF with the cells. In different 3T3 cell lines the magnitude of the increase was approximately proportional to the number of PDGF receptors per cell. A number of proteins phosphorylated at tyrosine in response to PDGF have been detected by two-dimensional gel electrophoresis. They include a pair of related 45 kilodalton phosphoproteins, a pair of related 43 kilodalton phosphoproteins and a 42 kilodalton phosphoprotein. Similar changes were noted when quiescent 3T3 cells were incubated with epidermal growth factor. Possibly, these phosphoproteins are primary substrates of the tyrosine protein kinases activated by the receptors for PDGF and epidermal growth factor, and are involved in physiological effects common to the two growth factors.

The transforming proteins of Rous sarcoma virus, Harvey sarcoma virus and Abelson virus contain tightly bound lipid

We have found that the transforming proteins of Rous sarcoma virus, Harvey sarcoma virus and Abelson virus all contain tightly bound lipid. This modification could play a role in the binding of these proteins to cellular membranes. The lipid associated with p60src, the transforming protein of Rous sarcoma virus, is located in the NH2-terminal domain of the polypeptide. This is the region of the protein that has been shown previously to participate in binding the protein to membranes. Two mature forms of p21, the transforming protein of Harvey sarcoma virus, contain lipid. Lipid is not, however, associated with newly synthesized p21. While mature p60src and p21 are bound to cellular membranes, the newly synthesized forms of these proteins are not. The posttranslational addition of lipid may therefore be the means by which these proteins acquire an affinity for membranes.

Four different classes of retroviruses induce phosphorylation of tyrosines present in similar cellular proteins.

Chicken embryo cells transformed by the related avian sarcoma viruses PRC II and Fujinami sarcoma virus, or by the unrelated virus Y73, contain three phosphoproteins not observed in untransformed cells and increased levels of up to four other phosphoproteins. These same phosphoproteins are present in increased levels in cells transformed by Rous sarcoma virus, a virus which is apparently unrelated to the three aforementioned viruses. In all cases, the phosphoproteins contain phosphotyrosine and thus may be substrates for the tyrosine-specific protein kinases encoded by these viruses. In one case, the site(s) of tyrosine phosphorylation within the protein is the same for all four viruses. A homologous protein is also phosphorylated, at the same major site, in mouse 3T3 cells transformed by Rous sarcoma virus or by the further unrelated virus Abelson murine leukemia virus. A second phosphotyrosine-containing protein has been detected in both Rous sarcoma virus and Abelson murine leukemia virus-transformed 3T3 cells, but was absent from normal 3T3 cells and 3T3 cells transformed by various other viruses. We conclude that representatives of four apparently unrelated classes of transforming retroviruses all induce the phosphorylation of tyrosines present in the same set of cellular proteins.

Regulation of Cell Growth and Transformation by Tyrosine-Specific Protein Kinases: The Search for Important Cellular Substrate Proteins

It is now well established that post-translational modification of vertebrate cell proteins can occur by phosphorylation at serine, threonine, and tyrosine. Many serine- and threonine-speciflc protein kinases and their substrates have been extensively characterized, and in several instances protein phosphorylation has been shown to play an important role in the regulation of cell metabolism through alteration of the properties of specific enzymes. Historically, evidence that modulation of protein function led to a significant change in metabolism often predated the discovery that the protein in question was a phosphoprotein and the isolation of the regulatory protein kinase. For example, increased glycogenolysis in liver slices, under conditions of adrenaline or glucagon treatment, was correlated with activation of the enzyme glycogen phosphorylase. The active form of glycogen phosphorylase was subsequently found to be a phosphoprotein, and the protein kinase responsible, phosphorylase kinase, was characterized (Krebs and Fischer 1956). The regulation of phosphorylase kinase itself by phosphorylation has since been elucidated in great detail (Cohen 1978).

6 Viral Oncogenes and Tyrosine Phosphorylation

Publisher Summary This chapter describes viral protein-tyrosine kinases and discusses their roles in viral transformation. For this, the properties of the individual viral protein-tyrosine kinases are described in the chapter and are contrasted to the cognate cellular enzymes that are encoded by the cellular genes homologous to the viral oncogenes. The chapter discusses a number of transformed cell types where altered tyrosine phosphorylation is evident and describes the general properties of protein-tyrosine kinases and their common structural features. In addition, the chapter includes a progress report on the identification of substrates for the viral protein-tyrosine kinases. The chapter discusses the way in which viral protein-tyrosine kinases differ from their cellular counterparts—enzymes that clearly coexist peaceably with normal cells. In the chapter, the properties of the viral enzymes are compared and contrasted to those of the growth factor receptor protein-tyrosine kinases, particularly because of the abnormal growth state of transformed cells.

Role of Tyrosine Phosphorylation in Malignant Transformation by Viruses and in Cellular Growth Control

Publisher Summary This chapter discusses the role of tyrosine phosphorylation in malignant transformation by viruses and in cellular growth control. Tyrosine protein-kinases appear to be essential cellular gene products. The regulated phosphorylation of tyrosine in proteins has been implicated in growth control and the organization of cytoskeleton in normal cells. The molecular basis for transformation by some acutely transforming retroviruses may involve unscheduled phosphorylation of cellular proteins on tyrosine. One indication of this is that cells transformed by these viruses have phosphotyrosine levels in protein five to ten fold higher than the parental untransformed cells. Moreover, the transforming proteins of these viruses have an associated tyrosine-specific protein-kinase activity. All acutely transforming retroviruses contain in their genomes sequences recognizable as having been derived from the genome of a normal cell. These cellular sequences are responsible for the oncogenic potential of these viruses, encoding at least part of their transforming proteins. The group of viruses whose transforming proteins have associated tyrosine protein-kinase activity can be subdivided into five classes representing five distinguishable types of cellular sequence. The onc genes corresponding to these cellular sequences are src, fps or fes, yes, ros, and abl . In addition to these five genes, evidence is mounting that the genes encoding certain growth-factor receptors may also specify tyrosine protein-kinases. There are strong indications that the epidermal growth factor receptor has intrinsic tyrosine protein-kinase activity.

Protein Phosphorylation at Tyrosine in Normal and Transformed Cells

Retroviruses incorporating each of the oncogenes v-src, v-fps (a chicken gene whose mammalian homologue is v-fes), v-abl, v-yes or v-fgr (apparently the yes gene with added actin sequences) share the ability to increase the content of phosphotyrosine in the proteins of cells they transform (reviewed [1,2]). The transforming proteins encoded by these five genes are active tyrosine protein kinases in vitro, so it is assumed that much of the increase in tyrosine phosphorylation observed in the transformed cells is catalyzed directly by the retroviral transforming proteins, although it is possible that additional, cellular, tyrosine protein kinases are activated. However, tyrosine protein kinase activation is not a universal correlate of transformation. Cells transformed by chemicals, some tumor oncogenes, DNA tumor viruses, and many retroviruses have “normal” low levels of phosphotyrosine in their proteins (3). Even some retroviruses whose oncogenes have considerable sequence homology to the v-src-family of oncogenes do not increase cellular protein phosphotyrosine content detectably. Notable among these are the viruses that contain v-mil (a chicken gene whose mammalian homologue is v-raf), v-ros, v-fms, and v-mos (4–9). Paradoxically, the v-ros product is an active tyrosine protein kinase when isolated by immunoprecipitation yet cells transformed by UR2 virus, which encodes a gag-ros fusion protein, have only slightly enhanced levels of phosphotyrosine (9; Sefton BM, personal communication).