pp60c-src variants containing lesions that affect phosphorylation at tyrosines 416 and 527.

The biological and biochemical properties of pp60c-src are regulated, in part, by phosphorylation at Tyr-416 and Tyr-527. The tyrosine kinase and transforming activities of pp60c-src are suppressed by phosphorylation at Tyr-527, whereas full activation of pp60c-src requires phosphorylation at Tyr-416. To test specifically the significance of the negatively charged phosphate moieties on these tyrosine residues, we have substituted the codons for both residues with codons for either Glu or Gln. A negatively charged Glu at position 527 was unable to mimic a phosphorylated Tyr at this position, and, in consequence, the mutated pp60c-src was activated and transforming. Similarly, substitution of Tyr-416 with Glu was unable to stimulate the activities of the enzyme. However, mutagenesis of Tyr-416 to Gln (to form the mutant 416Q) activated the kinase activity approximately twofold over that observed for wild-type pp60c-src. When introduced into the mutant 527F (containing Phe-527 instead of Tyr), the double mutant 416Q-527F exhibited weak transforming activity. This is in contrast to the other double mutants 416E-527F and 416F-527F, which were nontransforming. The biochemical basis by which 416Q activates pp60c-src is not understood but probably involves some local conformational perturbation. Deletion of residues 519 to 524 (RH5), a region previously shown to be necessary for association with middle-T antigen, led to loss of phosphorylation at Tyr-527 and activation of the enzymatic and focus-forming activities of pp60c-src. Hence, the sequences necessary for complex formation with middle-T antigen may also be required by the kinase(s) which phosphorylates Tyr-527 in vivo. This suggests that normal cells contain cellular proteins which are analogous to middle-T antigen and whose action regulates the activity of pp60c-src by controlling phosphorylation or dephosphorylation at residue 527.     

Regulation of the oncogenic activity of the cellular src protein requires the correct spacing between the kinase domain and the C-terminal phosphorylated tyrosine (Tyr-527).

Repression of the tyrosine kinase activity of the cellular src protein (pp60c-src) depends on the phosphorylation of a tyrosine residue (Tyr-527) near the carboxy terminus. Tyr-527 is located 11 residues C terminal from the genetically defined end of the kinase domain (Leu-516) and is therefore in a negative regulatory region. Because the precise sequence of amino acids surrounding Tyr-527 appears to be unimportant for regulation, we hypothesized that the conformational constraints induced by phosphorylated Tyr-527 may require the correct spacing between the kinase domain (Leu-516) and Tyr-527. In this report, we show that deletions at residue 518 of two, four, or seven amino acids or insertions at this residue of two or four amino acids activated the kinase activity and thus the transforming potential of pp60c-src. As is the case for the prototype transforming variant, pp60527F, activation caused by these deletions or insertions was abolished when Tyr-416 (the autophosphorylation site) was changed to phenylalanine. In comparison with wild-type pp60c-src, the src proteins containing the alterations at residue 518 showed a lower phosphorylation state at Tyr-527 regardless of whether residue 416 was a tyrosine or a phenylalanine. Mechanisms dealing with the importance of spacing between the kinase domain and Tyr-527 are discussed.     

Dephosphorylation or antibody binding to the carboxy terminus stimulates pp60c-src.

Phosphorylation of pp60c-src at Tyr-527, six residues from the carboxy terminus, has been implicated in regulation of the protein-tyrosine kinase activity of pp60c-src. Here we show that dephosphorylation of pp60c-src by phosphatase treatment in vitro caused a 10- to 20-fold increase in pp60c-src protein-tyrosine kinase activity. Binding of specific antibody to the region of pp60c-src which contains phosphotyrosine-527 also increased kinase activity. Each treatment increased phosphorylation of added substrates and of Tyr-416 within pp60c-src by a similar mechanism that involved altered interactions with ATP and increased catalytic rate. We suggest that the phosphorylated carboxy terminus acts as an inhibitor of the protein kinase domain of pp60c-src, unless its conformation is altered by either dephosphorylation or antibody binding. The antibody additionally stimulated the phosphorylation of forms of pp60c-src that had reduced gel mobility, much like those phosphorylated in kinase reactions containing pp60c-src activated by polyomavirus medium tumor antigen. These in vitro experiments provide models for the activation of pp60c-src in cells transformed by polyomavirus. We also show that autophosphorylation of pp60c-src at Tyr-527 occurs only to a very limited extent in vitro, even when Tyr-527 is made available for phosphorylation by treatment with phosphatase. This suggests that other protein-tyrosine kinases may normally phosphorylate Tyr-527 and regulate pp60c-src in the cell.     

Regulation of pp60c-src and its interaction with polyomavirus middle T antigen in insect cells.

High yields of soluble, biologically active pp60c-src and middle t antigen (MTAg) of polyomavirus were produced in insect cells, using a baculovirus expression system. In mammalian cells, pp60c-src undergoes a regulatory phosphorylation on Tyr-527 in vivo and is autophosphorylated on Tyr-416 in vitro. In insect cells, pp60c-src was phosphorylated primarily on Tyr-416, although Tyr-527 was detectable at a low level. A kinase-negative mutant of pp60c-src was not phosphorylated on either Tyr-527 or Tyr-416 in insect cells and thus is an excellent biochemical reagent to search for the regulatory kinase that usually phosphorylates Tyr-527 in mammalian cells. MTAg synthesized in insect cells was not phosphorylated on tyrosine residues in vivo or in vitro, suggesting that it did not associate with any endogenous tyrosine kinases. However, MTAg isolated from cells coinfected with viruses encoding both MTAg and pp60c-src was phosphorylated on tyrosine residues both in vivo and in vitro.     

Activation and suppression of pp60c-src transforming ability by mutation of its primary sites of tyrosine phosphorylation

pp60c-src is phosphorylated in vivo at tyrosine 527, a residue not present in pp60v-src (its transforming homolog), and not at tyrosine 416, its site of in vitro autophosphorylation. To test the hypothesis that tyrosine phosphorylation regulates pp60c-src biological activity, we constructed and studied pp60c-src mutants in which Tyr 527 and Tyr 416 were separately or coordinately altered to phenylalanine. Tyr----Phe 527 mutation strongly activated pp60c-src transforming and kinase activities, whereas the additional introduction of a Tyr----Phe 416 mutation suppressed these activities. Tyr----Phe 416 mutation of normal pp60c-src eliminated its partial transforming activity, which suggests that transient or otherwise restricted phosphorylation of Tyr 416 is important for pp60c-src function even though stable phosphorylation is not observed in vivo.     

Mutation of amino acids in pp60c-src that are phosphorylated by protein kinases C and A.

The product of the c-src proto-oncogene, pp60c-src, is phosphorylated at Ser-17 by cyclic AMP-dependent protein kinase A and at Ser-12 by calcium-phospholipid-dependent protein kinase C (when stimulated by 12-O-tetradecanoyl phorbol acetate). We tested the effects of Ser----Ala and Ser----Glu mutations at these sites in pp60c-src and in pp60c-src(F527) (a mutant whose transforming activities are enhanced by Tyr-527----Phe mutation) by transfecting single-, double-, and triple-mutant src expression plasmids into NIH 3T3 cells. Tryptic phosphopeptide analyses of the mutant proteins confirmed prior biochemical identifications of the phosphorylation sites and showed that neither separate nor coordinate mutations at Ser-12 and Ser-17 affected Tyr-416, Tyr-527, or Ser-48 phosphorylation or prevented mitosis-specific phosphorylations of either pp60c-src or pp60c-src(F527). Ser-12 mutation did not affect phosphorylation of the Ser-17-containing peptide, but mutation of Ser-17 significantly increased phosphorylation at Ser-12. Specific kinase activities (both with and without in vivo 12-O-tetradecanoyl phorbol acetate treatment) and the abilities of pp60c-src and pp60c-src(F527) to induce foci, transformed morphologies, and anchorage-independent growth were unaffected by any of the serine mutations. Thus, pp60c-src transforming activity in NIH 3T3 cells is relatively insensitive to phosphorylation at these sites, but there is a suggestion that Ser-17 phosphorylation may have a subtle regulatory effect.     

Cell transformation by pp60c-src mutated in the carboxy-terminal regulatory domain

We introduced two mutations into the carboxy-terminal regulatory region of chicken pp60c-src. One, F527, replaces tyrosine 527 with phenylalanine. The other, Am517, produces a truncated pp60c-src protein lacking the 17 carboxy-terminal amino acids. Both mutant proteins were phosphorylated at tyrosine 416 in vivo. The specific activity of the Am517 mutant protein kinase was similar to that of wild-type pp60c-src whereas that of the F527 mutant was 5- to 10-fold higher. Both mutant c-src genes induced focus formation on NIH 3T3 cells, but the foci appeared at lower frequency, and were smaller than foci induced by polyoma middle tumor antigen (mT). The wild-type or F527 pp60c-src formed a complex with mT, whereas the Am517 pp60c-src did not. The results suggest that one, inability to phosphorylate tyrosine 527 increases pp60c-src protein kinase activity and transforming ability; two, transformation by mT involves other events besides lack of phosphorylation at tyrosine 527 of pp60c-src; three, activation of the pp60c-src protein kinase may not be required for transformation by the Am517 mutant; and four, the carboxyl terminus of pp60c-src appears to be required for association with mT.     

Altered sites of tyrosine phosphorylation in pp60c-src associated with polyomavirus middle tumor antigen.

We characterized the tyrosine phosphorylation sites of free pp60c-src and of pp60c-src associated with the polyomavirus middle tumor antigen (mT) in transformed avian and rodent cells. The sites of tyrosine phosphorylation in the two populations of pp60c-src were different, both in vitro and in vivo. Free pp60c-src was phosphorylated in vitro at a single site, tyrosine 416. pp60c-src associated with mT was phosphorylated in vitro on tyrosine 416 and on one or more additional tyrosine residues located in the amino-terminal region of the molecule. Free pp60c-src in polyomavirus mT-transformed cells was phosphorylated in vivo on tyrosine 527. In contrast, pp60c-src associated with mT was phosphorylated in vivo on tyrosine 416 and not detectably on tyrosine 527. Thus, the in vivo phosphorylation sites of pp60c-src associated with mT in transformed cells are identical to those of pp60v-src, the Rous sarcoma virus transforming protein. The results suggest that altered phosphorylation of pp60c-src associated with mT may play a role in the enhancement of the pp60c-src protein kinase activity and in cell transformation by polyomavirus.     

Characterization of purified pp60c-src protein tyrosine kinase from human platelets.

Intact pp60c-src, the cellular homologue of the transforming protein of Rous sarcoma virus, was purified from human platelets. The purified fractions also contained small amounts of a 54-kDa proteolytic degradation product of pp60c-src. We investigated some of the biochemical and kinetic properties of pp60c-src protein tyrosine kinase. Maximum kinase activity occurred at pH 6.5 and required a mixture of 2 mM Mn2+/Mg2+ as divalent cations. The enzyme most strongly phosphorylated casein, followed by enolase and alcohol dehydrogenase. The Km value for ATP was 4 microM for substrate phosphorylation and for autophosphorylation. Using casein, we determined a Vmax for substrate phosphorylation by pp60c-src in the range of 1.9-3.4 nmol.min-1.mg-1. Since the Vmax value for the purified 54-kDa fragment of pp60c-src was also included in this value, we conclude that proteolytic degradation of a 6-kDa fragment from the N-terminus of pp60c-src did not affect its kinase activity. Tryptic phosphopeptide analysis identified Tyr-416 as the major autophosphorylation site. Preincubation of purified pp60c-src with ATP increased the amount of autophosphorylation accompanied by an increase in Vmax, whereas the Km values were not altered. Our data directly demonstrate that autophosphorylation at Tyr-416 exerts, in contrast to phosphorylation at Tyr-527, a positive regulatory effect on the pp60c-src kinase activity.     

Neoplastic transformation induced by an activated lymphocyte-specific protein tyrosine kinase (pp56lck).

The lck proto-oncogene encodes a lymphocyte-specific member of the src family of protein tyrosine kinases. Here we demonstrate that pp56lck is phosphorylated in vivo at a carboxy-terminal tyrosine residue (Tyr-505) analogous to Tyr-527 of pp60c-src. Substitution of phenylalanine for tyrosine at this position resulted in increased phosphorylation of a second tyrosine residue (Tyr-394) and was associated with an increase in apparent kinase activity. In addition, this single point mutation unmasked the oncogenic potential of pp56lck in NIH 3T3 cell transformation assays. Viewed in the context of similar results obtained with pp60c-src, it is likely that the enzymatic activity and transforming ability of all src-family protein tyrosine kinases can be regulated by carboxy-terminal tyrosine phosphorylation. We further demonstrate that overexpression of pp56lck in the murine T-cell lymphoma LSTRA as a result of a retroviral insertion event produces a kinase protein that despite wild-type primary structure is nevertheless hypophosphorylated at Tyr-505. Thus, control of normal growth in this lymphoid cell line may have been abrogated through acquisition of a posttranslationally activated version of pp56lck.     

Myristylation of pp60c-src is not required for complex formation with polyomavirus middle-T antigen.

Middle-T antigen (middle-T), the transforming gene product of polyomavirus, associates with several cellular tyrosine kinases, such as pp60c-src. Complex formation leads to kinase activation and is essential for cell transformation. Middle-T-associated as well as uncomplexed pp60c-src is predominantly found in the plasma membrane. We transfected mouse 3T3 fibroblasts with a mutated c-src gene (2Ac-src), allowing the expression of a protein containing alanine instead of glycine in position 2 of the primary translation product. Contrary to the wild-type c-src gene product, pp60c-src(2A) was not myristylated and accumulated in the cytoplasm instead of being transferred to cellular membranes. The mutant protein was able to associate with middle-T and was activated similarly to the wild-type c-src gene product. Both wild-type and 2A mutant protein were membrane associated upon complex formation with middle-T. This finding suggests that the putative carboxy-terminal membrane anchor sequence of middle-T is sufficient to hold middle-T-associated pp60c-src(2A) in the plasma membrane.     

Mutations around the NG59 lesion indicate an active association of polyoma virus middle-T antigen with pp60c-src is required for cell transformation.

The transforming activity of polyoma virus middle-T antigen is believed to be dependent on its ability to form a complex with the cellular tyrosine protein kinase, pp60c-src. This hypothesis is based on observations of mutants of middle-T which demonstrated a correlation between these two activities. To investigate further the significance of pp60c-src association in transformation by middle-T, a series of deletion and point mutants were constructed around the NG59 lesion since this region has been implicated in pp60c-src binding. Analysis of the middle-T variants revealed a complete correlation between the presence of associated activated pp60c-src and the ability to transform. Further, this ability of pp60c-src to associate with middle-T may depend on the presence of a beta-turn between amino acids 177 and 180. The results indicate the NG59 phenotype results from the introduction of an isoleucine residue between amino acids 177 and 178 rather than the transition mutation at 179. The mutant MG1 is a single point mutation (at residue 180) and represents the smallest change in the middle-T which abolishes both the transformating and kinase activity of middle-T. Taken together, the data suggest the region surrounding the NG59 lesion is involved in the formation of an active complex between middle-T and pp60c-src and strongly suggest that this association is an absolute requirement for polyoma virus-induced transformation.     

Alterations in pp60c-src accompany differentiation of neurons from rat embryo striatum.

Cultured neurons from rat embryo striatum were found to contain two structurally distinct forms of pp60c-src. The 60-kilodalton (kDa) form appeared similar to pp60c-src from cultured rat fibroblasts or astrocytes. The 61-kDa form was specific to neurons and differed in the NH2-terminal 18 kDa of the molecule. In undifferentiated neurons the predominant phosphorylated species of pp60c-src was the fibroblast form. Upon differentiation, a second phosphorylated form of pp60c-src was detected. This form had two or more additional sites of serine phosphorylation within the NH2-terminal 18-kDa region of the molecule, one of which was Ser-12. The specific protein-tyrosine kinase activity of the total pp60c-src population increased 14-fold, as measured by autophosphorylation, or 7-fold, as measured by phosphorylation of an exogenous substrate, as striatal neurons differentiated. This elevation in protein kinase activity occurred without a detectable decrease in Tyr-527 phosphorylation or increase in Tyr-416 phosphorylation. Our results support the idea that the expression of the neuron-specific form of pp60c-src and the increase in specific protein kinase activity may be important for neuronal differentiation.     

Peptide antibodies to the human c-fyn gene product demonstrate pp59c-fyn is capable of complex formation with the middle-T antigen of polyomavirus.

The c-fyn proto-oncogene is a member of a family of closely related genes of which c-src is the prototype. Using peptide antibodies which had been raised against sequences predicted to be specific for the human c-fyn gene product, the c-fyn protein was identified. It is a tyrosine kinase with apparent mol. wt of 59 kd that is also phosphorylated and myristylated. Like pp60c-src and pp62c-yes, pp59c-fyn is able to form a stable complex with middle-T antigen, the transforming protein of polyomavirus. The transformation-defective middle-T mutant NG59, which is unable to associate stably with pp60c-src does not associate with pp59c-fyn. In contrast to pp60c-src, complex formation with middle-T antigen does not lead to a significant increase in the tyrosine kinase activity of pp59c-fyn. These findings lead us to suggest that middle-T mediated transformation may be a consequence of the deregulation of several members of the src-family of protein tyrosine kinases.     

Regulation by the autophosphorylation site in overexpressed pp60c-src.

We show that overexpressed pp60c-src is phosphorylated at Tyr-416 and has increased specific kinase activity when isolated from cells incubated with vanadate, a tyrosine phosphatase inhibitor. This supports the hypothesis that transient Tyr-416 phosphorylation modulates the activity of overexpressed pp60c-src in vivo. Mutagenesis indicates that Tyr-416 modulates pp60v-src activity as well.     

Substitution of Ser-17 of pp60c-src: biological and biochemical characterization in chicken embryo fibroblasts.

pp60c-src is phosphorylated mainly on Ser-17 and Tyr-527 in vivo. In this study, we examined the effect of the phosphorylation of Ser-17 on the properties of pp60c-src by introducing Rous sarcoma virus variants carrying pp60c-src in which Ser-17 had been substituted, into chicken embryo fibroblasts. The Ala-17 substitution in wild-type pp60c-src and pp60c-src carrying Phe-527 caused a two- to threefold elevation in the kinase activity in vitro of these proteins; the former variant resulted in no morphological changes of infected cells, whereas the latter variant transformed chicken embryo fibroblasts. Since the substitution of Tyr-527 per se has been reported to activate pp60c-src, these results suggest that the abolishment of the phosphorylation of Ser-17 does not affect noticeably the properties of pp60c-src in chicken embryo fibroblasts.     

The complex of polyoma virus middle-T antigen and pp60c-src.

We have recently proposed that the transforming protein of polyoma virus, middle-T antigen, forms a complex with pp60c-src. Here we provide additional evidence for the existence of the complex using both monoclonal antibodies specific for middle-T and antibodies raised against synthetic peptides corresponding to sequences from both middle-T and pp60c-src. The complex was retained during partial purification of middle-T and was stable to incubation under various conditions. A survey of a number of mutants of middle-T antigen showed that there was a complete correlation between the ability of middle-T to accept phosphate in the in vitro kinase reaction and the presence of a middle-T: pp60c-src complex. This result is in accord with our hypothesis that middle-T itself is not a protein kinase but rather that pp60c-src phosphorylates middle-T. All mutant forms of middle-T antigen capable of transformation had associated pp60c-src. The middle-T of two non-transforming mutants (hr-t mutants) did not have associated pp60c-src, whereas other non-transforming middle-T species did associate with pp60c-src. We propose that the complex plays an essential role in transformation by polyoma virus, but that the existence of the complex per se may not be sufficient.     

Structural differences between repressed and derepressed forms of p60c-src.

The kinase activity of p60c-src is derepressed by removal of phosphate from Tyr-527, mutation of this residue to Phe, or binding of a carboxy-terminal antibody. We have compared the structures of repressed and active p60c-src, using proteases. All forms of p60c-src are susceptible to proteolysis at the boundary between the amino-terminal region and the kinase domain, but there are several sites elsewhere that are more sensitive to trypsin digestion in repressed than in derepressed forms of p60c-src. The carboxy-terminal tail (containing Tyr-527) is more sensitive to digestion by pronase E and thermolysin when Tyr-527 is not phosphorylated. The kinase domain fragment released with trypsin has kinase activity. Relative to intact p60c-src, the kinase domain fragment shows altered substrate specificity, diminished regulation by the phosphorylated carboxy terminus, and novel phosphorylation sites. The results identify parts of p60c-src that change conformation upon kinase activation and suggest functions for the amino-terminal region.     

Tyrosine phosphorylation regulates the biochemical and biological properties of pp60c-src

To investigate the importance of tyrosine phosphorylation in the regulation of pp60c-src, we have substituted phenylalanine for tyrosine at positions 416, 519, and 527. Cells expressing the 527 or the 519/527 mutant but not the 416 or the 519 mutant were morphologically transformed, grew in soft agar, and formed foci. In addition, the 527 and 519/527 mutants had elevated kinase activities in vitro. Modifying Tyr 416 to phenylalanine in the 527 or the 519/527 mutants only partially inhibited their kinase activities yet abolished their ability to induce focus formation and promote growth in soft agar. These results suggest that two events must occur to activate the full transforming potential of pp60c-src: hypophosphorylation at Tyr 527 and hyperphosphorylation at Tyr 416.     

Mapping of the amino-terminal half of polyomavirus middle-T antigen indicates that this region is the binding domain for pp60c-src.

The majority of the carboxy-terminal half of polyomavirus middle-T antigen has been variously mutated and, with the exception of the putative membrane-binding domain (amino acids 394 to 415), was found to be largely dispensible for the transforming activity of the protein. A comparison of the small-T antigen amino acid sequences (equivalent to the region of middle-T encoded by exon 1) of simian virus 40, BK virus, polyomavirus, and a recently described hamster papovavirus highlighted regions of potential interest in mapping functions to the amino-terminal half of polyomavirus middle-T antigen. The regions of interest include amino acids 168 to 191 (previously investigated by this group [S. H. Cheng, W. Markland, A. F. Markham, and A. E. Smith, EMBO J. 5:325-334, 1986]), two cysteine-rich clusters (amino acids 120 to 125 and 148 to 153), and amino acids 92 to 117 (within the limits of the previously described hr-t mutant, SD15). Point mutations, multiple point mutations, and deletions were made by site-specific and site-directed mutagenesis within the cysteine-rich clusters and residues 92 to 117. Studies of the transforming ability of the altered middle-T species demonstrated that this activity is highly sensitive to amino acid changes. All four regions (as defined above) within the amino-terminal half of middle-T have now been studied in detail. The phenotype of the mutants is predominantly transformation defective, and the corresponding variant middle-T species are characterized by being either totally or severely handicapped in the ability to associate actively with pp60c-src. Whether the mutations affect the regions of interaction between middle-T and pp60c-src or simply interfere with the overall conformation of this domain is not known. However, there would appear to be a conformational constraint on this portion of the molecule with regard to its interaction with pp60c-src and by extension to the ability of the middle-T species to transform.