Specific TPA-induced protein phosphorylations in cultured myotubes

Cell Biology

International

SPECIFIC

TPA-INDUCED

M. BouchC, Istituto

di

mptly

Differentiated respond

S.Adamo Istologia

to

PROTEIN and

Reports,

Vol. 7, No. 3, March

PHOSPHOHYLATIONS

M. Mollr~aro ed Embriologla

myotubes 12-O-tetradecanoyl

1"

generale, culture

pro phor-

bol 13-acetate (TPA) with reversible dedif ferentiative and transformation-like than reentering the cell cycle ges , without (1,Z). It has been reported that the earliest effects of TPA include changes in Ca++ fluxes and cyclic nucleotide levels, within few minutes of its administration to myogenic cells (3,4). We uluestigated whether alterations of the phosphorylation of endogenous substrates were evidentiable as a" early response to TPA treatment. Chick embryo myotubes were cultured described (2) and 96 h cultures, predominantly containing multinucleated myotubes, were treated with 100 nM TPA, O,l% DMSO, or with 0.1% DMSO alone for 1 h. During this period 3zPi (orthophosphoric acid 0.1 mCl/ml) was added for the indicated intervals. As shown in Fig. 1 TPA induces fold increase 1" the incorporatiorl 3zPii" both the acid soluble and insoluble fraction of myotubes.

IN

as

CULTURED

UniversitB For treated for 15'

1983

189

MYOTUBES

dl SDS-PAGE with TPA with 32Pi.

Roma

-Italy

analysis or DMSO

the cells and incubated

were

The cells were washed with PBS, scraped and precipitated with 10% TCA at 4OC; the pellet was incubated with 5% TCA at 95OC for 5', washed twice with cold 5% TCA and extracted 3 times with 99% methanol. The residue was solubilized in sample buffer (5) and aliquots containing equal amounts of radioactivity were layered on slab gels (3% stacking, 10% separatory gel) prepared according to Laemmli (5). After electrophoresis the gels were exposed for autoradio@-Wv

. Fig. 2. SDS-PAGE ted with

Autoradiograpy of myotubes DMSO (left

or TPA (right lane) 151, and incubated O.SmCi/ml 3zPi in The arrow me time. cates the position 26,000 polypeptlde.

time

a 2of the acid

of trea lane) for with the saindiof

Mr

Fig. 2 shows that TPA specifically and strongly stimulates the phosphorylation of a polypeptide of Mr 26,000 and, to a lesser extent, that of polypeptide of Mr 33,000. Such stimulation of the phosphorylation of specific polypeptides occurs after TPA treatments of different lenght of time (15'-10 h). Similar effects are Fig. 1 Time course of "Pi incorporation in control (w or TPA treated (-1 myotubes.CelIs were extracted twice with 5% PCA I" the cold and the acid insoluble residue was solubillzed with IN NaOH.Aliquots tely vity.

1. 2. 3. 4. 5. *.lhis trol

of the neutralized

two

fractions and counted

were appropria for radloactl

not "on

detectable 1" cells treated with the tumor promoter TPA analog 4CZ -PDD. The data reported Indicate that TPA Induces a generalized increase I" the incorporation of phosphate but it also induces a selective stimulation of the phospho-

rylation of specific substrates as early as 15' of treatment. These early modifications may be involved in the mechanism by which TPA Induces the later dedifferentlatlve and transformation-like changes.*

Cohen R Pacific1 M Hublnstei" N Blehl J & Holtzer H (1977) Nature &6, 538-540. Cossu G Paciflci M Adamo S Bouche M & Mollnaro M (1982) Differentlatio" 21, 62-65. Schimmel S D & Hallam T (1980) Blochem Biophys Hes Comm 92, 624-030. Grotendorst G W & Schimmel S D (1980) Biochem Blophys Res Comm 91, 301-307. Laemmli U (1970) Nature 277, 680-685. work was supported by grant, No. 81.Oi415.06 of the C.N.R. finalized project "Conof tumor growth". Received: 15th Decrmber 1982. Accepted: 4th January 1983

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