PROTEIN PHOSPHORYLATION AND INTRACELLULAR SIGNALING 
Edwin G. Krebs, M.D., Senior Investigator 
The primary focus of this laboratory has been 
elucidation of the role of protein phosphorylation 
reactions in signal transduction. To this end the 
regulatory properties of several messenger-depen- 
dent and messenger-independent protein serine ki- 
nases have been investigated. In addition, limited 
studies have been carried out on protein serine 
phosphatases. A new area of interest is a heretofore 
neglected group of enzymes, the protein tyrosine 
phosphatases. 
I. Activation of Protein Kinases in Amphibian and 
Echinoderm Oocytes. 
In experiments directed toward examining the 
role of protein phosphorylation in the meiotic cell 
cycle, a synthetic peptide, Arg-Arg-Leu-Ser-Leu-Arg- 
Ala (the structure of which is based on a phosphor- 
ylated sequence in ribosomal protein S6), was em- 
ployed as a probe for measuring protein serine 
kinase activity in Xenopus laevis oocytes induced to 
mature by insulin or progesterone. Insulin elicited 
an early (20-30 min) 3-fold stimulation of S6 pep- 
tide-phosphorylating activity that was not evident 
with progesterone. However, both hormones pro- 
duced a delayed 7- to 12-fold stimulation of S6 pep- 
tide-phosphorylating activity at the time of germinal 
vesicle breakdown (GVBD). The results of DEAE- 
Sephacel, Sephacryl S-200, TSK-400, and heparin- 
Sepharose chromatographic fractionation experi- 
ments implied that a common 86 peptide kinase 
was activated as a consequence of short- and long- 
term insulin exposure, as well as in long-term pro- 
gesterone treatment of oocytes. Further work on 
the pathways involved in the activation of the 56 
peptide kinase is being undertaken. 
In addition to examining protein kinase activa- 
tion in amphibian oocytes, studies were also under- 
taken using starfish (sea star) oocytes, which are 
abundant in Puget Sound and readily available to 
this laboratory. (In this organism, 1-methyladenine 
is the natural hormone that induces maturation, in- 
stead of progesterone, as in vertebrates.) Five acti- 
vated protein kinases were detectable in soluble ex- 
tracts from maturing, as compared with immature, 
sea star oocytes. These kinases could be distin- 
guished on the basis of the time courses of their ac- 
tivation after exposure of the oocytes to 1-methyl- 
adenine, their substrate specificities, and their 
chromatographic properties. A histone HI kinase 
(HHIK; 110,000) underwent maximal activation 
near the time of 1-methyladenine-induced GVBD. 
When myelin basic protein (MBP) was used as a 
substrate, HHIK and two additional kinases (MBPK- 
I and MBPK-II) were detectable. MBPK-II (M^ 
110,000) was fully activated at the time of GVBD, 
whereas peak activation of MBPK-I (M^ 45,000) oc- 
curred after this event. Two "ribosomal protein ki- 
nases" (S6K-I and S6K-II) underwent activation 
post-GVBD. The HHIK and MBPK-II activities both 
declined prior to the emission of the first polar 
body (i.e., meiotic cell division), but the MBPK-I, 
S6K-I, and S6K-II activities remained elevated dur- 
ing this time. Developments in other laboratories 
during the past year make it seem probable that 
HHIK is a component of the maturation-promoting 
factor (MPF). 
II. Casein Kinase II. 
This laboratory has a special interest in a messen- 
ger-independent protein kinase known as casein ki- 
nase II (CK-II), which is activated when cells are ex- 
posed to growth factors. CK-II is found in the 
nucleus, as well as in the cytoplasm, and in this 
connection it is of considerable interest to find that 
the kinase catalyzes the phosphorylation of a num- 
ber of nuclear oncoproteins. It was shown, for ex- 
ample, that cellular myc-encoded proteins can 
serve as substrates for phosphorylation by purified 
CK-II in vitro and that this phosphorylation is re- 
versible. One- and two-dimensional mapping exper- 
iments demonstrated that the major phos- 
phopeptides from in vivo phosphorylated Myc 
corresponded to the phosphopeptides produced 
from Myc phosphorylated in vitro by CK-II. In addi- 
tion, synthetic peptides with sequences corre- 
sponding to putative CK-II phosphorylation sites in 
Myc were subject to multiple, highly efficient phos- 
phorylations by CK-II and could act as competitive 
inhibitors of CK-II phosphorylation of Myc in vitro. 
It was postulated that CK-II-mediated phosphoryla- 
tion of Myc plays a role in signal transduction to 
the nucleus. 
III. Activation of Protein Serine Phosphatase 1 by 
Growth Factors. 
Although most workers have concentrated on 
protein kinases as the targets for regulation by 
Continued 
81 
