expressed in fibroblasts displayed two- to fourfold 
higher specific activity than insert-minus pp60'^"*'^'^. 
In addition, the c-src^ protein expressed in fibro- 
blasts was not phosphorylated at the serine resi- 
due previously shown to be phosphorylated in 
pp60'^"'''^'^^ synthesized in neurons. These results 
indicated that the six-amino acid insertion in 
pp60'^'^'^'^"'" was sufficient to cause an increase in the 
specific activity of the c-src protein. Chicken em- 
bryo fibroblasts that expressed high levels of this 
protein displayed an altered morphology compared 
with cells that overexpressed pp60'^'^'^'^ (slightly 
more refractile and less contact-inhibited). In addi- 
tion, the c-src(^) overexpressor cells formed larger 
colonies in soft agar than the insert-minus c-src 
overexpressors and displayed higher levels of tyro- 
sine phosphorylation. Thus the six-amino acid in- 
sertion outside of the catalytic domain of pp60'^'^'^'^ 
modulates the kinase activity of this protein, caus- 
ing an increase in its kinase activity in vitro and in 
vivo. Current studies are focused on localization of 
pp60'^'^'^'^^ in neural tissues and elucidation of the 
functional role of this protein in neurons. 
III. Modulation of the Functional Activity of the 
c-src Protein through Site-directed Mutagenesis. 
Several lines of evidence indicate that sequences 
within the amino-terminal half of pp60'^'^'^'^ serve to 
regulate the catalytic activity of this protein tyrosine 
kinase. To examine how mutations outside of the 
catalytic domain of src influence the fianctional ac- 
tivity of the molecule, Larry Fox, Susan Nemeth, 
and Mike DeMarco have constructed and analyzed 
mutant forms of the protein containing single- 
amino acid substitutions and deletions throughout 
the amino-terminal half of the molecule. Mutations 
within the A homology box (residues 88-138, 
which are shared with seven src-related kinases, -y- 
phospholipase C, brain a-spectrin, and the v-crk 
oncogene) caused an activation of the kinase activ- 
ity and the transforming potential of the c-src pro- 
tein. Cells expressing elevated levels of these mu- 
tant proteins possessed elevated levels of tyrosine 
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phosphorylation, produced higher levels of plas- 
minogen activator, formed larger colonies in soft 
agar, and showed a more refractile and transformed 
cell morphology. The degree of activation corre- 
lated with the extent of the mutation, with single- 
amino acid substitutions causing the most subtle 
activation, and deletions of up to 75 amino acids, 
the strongest activation. These results suggest that 
sequences within this region of pp60'^'*'^'^ interact 
with the catalytic domain to maintain the protein in 
a low-activity state. Alterations within this region 
appear to relax this interaction and cause an activa- 
tion of c-src protein kinase activity. Mutations 
within the A box region have been combined with 
mutations in the B homology box and with muta- 
tions within the carboxyl-terminal half of src; syner- 
gistic, additive, or suppressive interactions between 
these domains have been examined. The analysis of 
these mutations is complicated; however, the 
following conclusions can be drawn: 1) Sequences 
within the B box [residues 148-190, which are 
shared with src-related tyrosine kinases, the 
GTPase-activating protein (GAP) of the ras protein, 
and 7-phospholipase C] are not necessary for main- 
tenance of the high kinase activity of all activated 
variants of c-src. 2) Elimination of the B box caused 
a severe restriction in the phosphorylation of most 
substrates in vivo (without affecting in vitro kinase 
activity, or the phosphorylation of several sub- 
strates in vivo, e.g., p36, calpactin). 3) Phosphory- 
lation of tyrosine 416 is necessary for full expres- 
sion of the biological activity of all activated forms 
of c-src, yet is not necessary for expression of a high 
catalytic activity of all transforming variants of c-src. 
These studies strongly suggest that sequences 
within the B box and phosphorylation of tyrosine 
416 affect the expression of the biological activity of 
the src protein in subtle ways that do not directly 
affect the catalytic activity per se. This work was 
also supported by grants from the National Cancer 
Institute. 
Dr. Brugge is also Professor of Microbiology at 
the University of Pennsylvania School of Medicine. 
Books and Chapters of Books 
Golden, A., and Brugge, J.S. 1988. The cellular and viral src gene. In The Oncogene Handbook (Reddy, E.E, 
Skalka, A.M., and Curran, T). Amsterdam: Elsevier Science, vol 7, pp 149-173. 
Continued 
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