P85 gag-mos encoded by ts110 Moloney murine sarcoma virus has an associated protein kinase activity

239

The v-at-' transforming protein P160' abl and the P210c-abl gene product of the translocated c-abl gene in Philadelphia chromosome-positive chronic myelogenous leukemia cells have tyrosine-specific protein kinase activity. Under similar assa) conditions the normal c-abl gene products, murine P150,-abl and human P145c-abl, lacked detectable kinase activity. Reaction conditions were modified to identify conditions which would permit the detection of c-abl tyrosine kinase activity. It was found that the Formalin-fixed Staphylococcus aureus formerly used for immunoprecipitation inhibits in vitro abl kinase activity. In addition, the sodium dodecyl sulfate and deoxycholate detergents formerly used in the cell lysis buffer were found to decrease recovered abl kinase activity. The discovery of assay conditions for c-abl kinase activity now makes it possible to compare P150c-abl and P145c.abl kinase activity with the altered abl proteins P160'-jabl and P210c-abl. Although all of the abl proteins have in vitro tyrosine kinase activity, they differ in the way they utilize themselves as substrates in vitro. Comparison of in vitro and in vivo tyrosine phosphorylation sites of the abl proteins suggests that they function differently in vivo. The development of c-abl kinase assay conditions should be useful in elucidating c-abl function.

Section: P145c-ah" Isolated From Ph'-positive K562 Cells (Data Notmentioning

confidence: 99%

Detection of c-abl tyrosine kinase activity in vitro permits direct comparison of normal and altered abl gene products.

Konopka¹,

Witte²

1985

239

“…Nevertheless, we cannot completely rule out the possibility that the c-abl P210 tyrosine kinase catalytic domain actually has dual specificity or that the serine phosphorylation is a result of the additional, unidentified amino-terminal sequences in c-abl P210. It has been reported that the P85gag-mos protein of the tsllO Moloney murine sarcoma virus has an associated serine and threonine kinase activity in vitro (13). Normal murine and human c-abl proteins are phosphorylated on serine in vivo.…”

mentioning

confidence: 99%

Activation of the c-abl oncogene by viral transduction or chromosomal translocation generates altered c-abl proteins with similar in vitro kinase properties.

Davis¹,

Konopka²,

Witte³

1985

153

The v-abl protein of Abelson murine leukemia virus is a tyrosine-specific kinase. Its normal cellular homolog, murine c-abl, does not possess detectable tyrosine kinase activity in vitro. Previously, we have detected tyrosine kinase activity in vitro for an altered c-abl gene product (c-abl P210) in the K562 human chronic myelogenous leukemia cell line. The expression of this variant c-abl gene product correlates with chromosomal translocation and amplification of the c-abl gene in K562 cells. Like v-abl, c-abl P210 is a fusion protein containing non-abl sequences near the amino terminus of c-abl. We compared the in vitro tyrosine kinase activity of c-abl P210 with that of wild-type murine v-abl. The remarkable similarities of these two proteins with respect to cis-acting autophosphorylation, trans-acting phosphorylation of exogenous substrates, and kinase inhibition, using site-directed abl-specific antisera, suggested that c-abl P210 could function similarly to v-abl in vivo. In addition, c-abl P210 possessed an associated serine kinase activity in immunoprecipitates. The serine kinase activity was not inhibited by site-directed, abl-specific antisera that inhibit the tyrosine kinase activity, suggesting that the serine kinase activity is not an intrinsic property of c-abl P210. Thus, the activation of the c-abl gene in a human leukemia cell line may have functional consequences analogous to activation of the c-abl gene in Abelson murine leukemia virus.

“…Since the function of the v-mos gene product has yet to be determined, neither its original role as a c-mos gene product nor its method of transformation when expressed by a retrovirus is known (12,27,35). Our work delineates the N-and C-terminal regions of the mos gene requisite for biological activity and consequently the sequences which are indispensable for an active v-mos gene product.…”

Section: Methodsmentioning

confidence: 94%

“…The v-mos gene product is one of several oncogenic proteins which show homology to the bovine cAMPdependent protein kinase but possess no specific tyrosine kinase activity (3,27). There are several specific regions within the v-rnos gene product which show conservation of residues with the protein kinase (Fig.…”

Section: Methodsmentioning

confidence: 99%

Biologically active mutants with deletions in the v-mos oncogene assayed with retroviral vectors.

Bold¹,

Donoghue²

1985

We have constructed retroviral expression vectors by manipulation of the Moloney murine leukemia virus genome such that an exogenous DNA sequence may be inserted and subsequently expressed when introduced into mammalian cels. A series of N-terminal deletions of the v-mos oncogene was constructed and assayed for biological activity with these retroviral expression vectors. The results of the deletion analysis demonstrate that the region of p37S coding region upstream of the third methionine codon is dispensable with respect to transformation. However, deletion mutants of v-mos which allow initiation of translation at the fourth methionine codon have lost the biological activity of the parental v-mos gene. Furthermore, experiments were also carried out to define the C-terminal limit of the active region of p37"OS by the construction of premature termination mutants by the insertion of a termination oligonucleotide. Insertion of the oligonucleotide just 69 base pairs upstream from the wild-type termination site abolished the focus-forming ability of v-mos. Thus, we have shown the N-terminal limit of the active region of p37I to be between the third and fourth methionines, while the C-terminal limit is within the last 23 amino acids of the protein.