Molecular cloning, expression and characterization of the human serine/threonine kinase Akt-3.

Akt (also known as PKB or RAC-PK) is an intracellular serine/threonine kinase involved in regulating cell survival. Although this makes it a promising target for the discovery of drugs to treat human cancer, a complicating factor may be the role played by Akt in insulin signalling. Two human isoforms, Akt-1 and Akt-2, have been described previously and a third isoform has been identified in rats (here termed Akt-3, but also called RAC-PK-gamma or PKB-gamma). We describe the identification of the corresponding human isoform of Akt-3. The gene encoding human Akt-3 was localized to chromosome 1q43-44. The predicted protein sequence is 83% identical to human Akt-1 and 78% identical to human Akt-2, and contains a pleckstrin homology domain and a kinase domain. In contrast to the published rat Akt-3 isoform, human and mouse Akt-3 also possess a C-terminal 'tail' that contains a phosphorylation site (Ser472) thought to be involved in the activation of Akt kinases. In addition to phosphorylation of Ser472, phosphorylation of Thr305 also appears to contribute to the activation of Akt-3 because mutation of both these residues to aspartate increased the catalytic activity of Akt-3, whereas mutation to alanine inhibited activation. Akt-3 activity could be inhibited by the broad spectrum kinase inhibitor staurosporine and by the PKC inhibitor Ro 31-8220, but not by other PKC or PKA inhibitors tested. Although Akt-3 is expressed widely, it is not highly expressed in liver or skeletal muscle, suggesting that its principle function may not be in regulating insulin signalling. These observations suggest that Akt-3 is a promising target for the discovery of novel chemotherapeutic agents which do not interfere with insulin signalling.

Inhibition of the Raf-1 kinase by cyclic AMP agonists causes apoptosis of v-abl-transformed cells.

Here we investigate the role of the Raf-1 kinase in transformation by the v-abl oncogene. Raf-1 can activate a transforming signalling cascade comprising the consecutive activation of Mek and extracellular-signal-regulated kinases (Erks). In v-abl-transformed cells the endogenous Raf-1 protein was phosphorylated on tyrosine and displayed high constitutive kinase activity. The activities of the Erks were constitutively elevated in both v-raf- and v-abl-transformed cells. In both cell types the activities of Raf-1 and v-raf were almost completely suppressed after activation of the cyclic AMP-dependent kinase (protein kinase A [PKA]), whereas the v-abl kinase was not affected. Raf inhibition substantially diminished the activities of Erks in v-raf-transformed cells but not in v-abl-transformed cells, indicating that v-abl can activate Erks by a Raf-1-independent pathway. PKA activation induced apoptosis in v-abl-transformed cells while reverting v-raf transformation without severe cytopathic effects. Overexpression of Raf-1 in v-abl-transformed cells partially protected the cells from apoptosis induced by PKA activation. In contrast to PKA activators, a Mek inhibitor did not induce apoptosis. The diverse biological responses correlated with the status of c-myc gene expression. v-abl-transformed cells featured high constitutive levels of expression of c-myc, which were not reduced following PKA activation. Myc activation has been previously shown to be essential for transformation by oncogenic Abl proteins. Using estrogen-regulated c-myc and temperature-sensitive Raf-1 mutants, we found that Raf-1 activation could protect cells from c-myc-induced apoptosis. In conclusion, these results suggest (i) that Raf-1 participates in v-abl transformation via an Erk-independent pathway by providing a survival signal which complements c-myc in transformation, and (ii) that cAMP agonists might become useful for the treatment of malignancies where abl oncogenes are involved, such as chronic myeloid leukemias.

Distinctive regulation of v-Src-associated phosphatidylinositol 3-kinase during PC12 cell differentiation.

In chicken embryo fibroblasts, the binding of v-Src to PtdIns 3-kinase requires Src homology domains, SH3, SH2 and the SH1 or kinase domain, which induces the cytoskeletal disruption associated with fibroblast transformation. In the rat phaeochromocytoma PC12 cell line, v-Src has a different effect on the cytoskeleton, inducing neurite extension rather than cytoskeletal disruption. Here we show that v-Src-induced neurite outgrowth is suppressed by the selective PtdIns 3-kinase inhibitor LY294002, suggesting that this effect of v-Src in PC12 cells also requires the activity of the lipid kinase. However, in contrast with chicken embryo fibroblasts, the association of PtdIns 3-kinase with v-Src in PC12 cells is delayed until several hours after activating the v-Src tyrosine kinase. Furthermore the v-Src-associated p85 regulatory subunit of PtdIns 3-kinase is not phosphorylated on tyrosine in PC12 cells and associates only weakly with isolated v-Src homology domains (SH3/SH2) in a Src kinase-independent manner. However, p85 and v-Src both associate with an unidentified protein (of molecular mass approx. 68 kDa; termed p68), which becomes tyrosine phosphorylated concomitantly with the association of both p85 and PtdIns 3-kinase with v-Src in PC12 cells. Thus we conclude that the mode of regulation of v-Src-associated PtdIns 3-kinase is cell-context-dependent and that p68 might act as an adaptor protein to mediate the association of p85 and v-Src in PC12 cells. The different regulation of PtdIns 3-kinase in PC12 and in chicken embryo fibroblasts in response to v-Src activity might reflect the different cytoskeletal rearrangements induced by this oncoprotein in the two cell types.

Cooperation of Src homology domains in the regulated binding of phosphatidylinositol 3-kinase. A role for the Src homology 2 domain.

Fibroblasts transformed by the v-Src oncoprotein exhibit elevated activity of the enzyme phosphatidylinositol 3'-kinase (PI 3-kinase), which binds to, and is activated by, a wide range of receptor tyrosine kinases as well as v-Src and transforming polyoma middle T/c-Src complexes. Here we consider the role of the v-Src homology (SH) domains, SH3 and SH2, and the tyrosine kinase catalytic domain, in the stimulation of v-Src-associated PI 3-kinase activity in response to rapid activation of the oncoprotein. As shown by others, we find that the v-Src SH3 domain tightly binds the PI 3-kinase p85 regulatory subunit in normal growing chicken embryo fibroblasts. However, we also find that in transformed cells there is additional efficient binding of PI 3-kinase to the v-Src SH2 domain in a catalytically active form. Furthermore, the binding of p85 to the SH2 domain, which is almost undetectable in quiescent cells, is rapidly stimulated upon activation of temperature-sensitive v-Src and consequent cell cycle entry, demonstrating that binding is a target for regulation. We also show that v-Src-associated PI 3-kinase differs considerably from PDGF receptor-associated enzyme by a different mode of binding, a lack of substantial allosteric activation, and a dependence on the tyrosine kinase activity of v-Src. The rapidly induced binding and activation of PI 3-kinase thus provides sensitive regulation of recruitment of PI 3-kinase to its substrates and into other signaling complexes at the cell membrane, which involves all the Src homology domains.

Mutations in v-Src SH3 and catalytic domains that jointly confer temperature-sensitive transformation with minimal temperature-dependent changes in cellular tyrosine phosphorylation.

We have analyzed two functionally significant amino acid alterations encoded by the temperature-sensitive (ts) v-src mutant of Rous sarcoma virus, LA32. The G-to-V change at residue 300 in the catalytic domain nonconditionally impairs morphological transformation, in vitro kinase activity, in vivo tyrosine phosphorylation, and the cytoskeletal association of v-Src while rendering anchorage- and serum-independent growth ts. The R-to-P mutation in the SH3 domain subtly enhances morphological transformation but has no phenotype if the catalytic domain is inactivated. In the presence of the G-300-to-V mutation, this SH3 domain lesion does not affect v-Src in vitro kinase activity and cytoskeletal association, but it nonconditionally enhances cellular tyrosine phosphorylation and restores morphological transformation at the permissive temperature only. This ability to induce a ts transformed morphology, in concert with nonconditional elevations of cellular phosphotyrosine, suggest that a subset of v-Src targets that are crucial to transformation may be affected in ts fashion by the SH3 mutation. Consistent with this, we find that the R-107-to-P mutation confers ts activity and tyrosine phosphorylation on the SH3-binding enzyme phosphatidylinositol 3'-kinase. Thus, both the SH3 and catalytic domain mutations in LA32 have some ts attributes and they cooperate in determining the mutant's behavior. The ts SH3 mutation is unique and offers the potential for deeper understanding of the function of this domain.

Functions of the v-Src protein tyrosine kinase.

The peripheral non-receptor tyrosine kinase oncoprotein, v-Src, has pleiotropic effects. It is a mitogen for quiescent cells, substituting for both competence and progression factor-mediated signals but it also induces cellular morphological transformation. We are dissecting the activities of v-Src by studying mutant proteins, including those with temperature sensitive (ts) effects, in different cellular backgrounds. Activation of a ts v-Src kinase rapidly increases activity of both the transcription factor, AP-1, and MAP kinase, an enzyme that enhances AP-1 activity by both phosphorylation of c-Jun and increased c-fos transcription; the relative contribution of these two events depends on the cells in which v-Src is expressed. Transient early AP-1 activation requires proper location of v-Src at the cell periphery and it is essential for mitogenesis. It is not, however, sufficient for entry into S-phase, there being a second need for v-Src later in G1. Transformation by v-Src does not require AP-1 activation but seems linked to events at the cell periphery, notably phosphorylation of proteins that bind to the v-Src SH3 domain such as the p85 subunit of PI-3 kinase.

Haemopoietic stem cell development to neutrophils is associated with subcellular redistribution and differential expression of protein kinase C subspecies.

Multipotential FDCP-Mix A4 (A4) cells can be induced either to self-renew or to differentiate and develop into mature neutrophils in liquid culture, depending on the haemopoietic growth factors with which they are cultured. When cultured in low concentrations of interleukin 3 (IL-3, 1 unit/ml)) plus Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) and Granulocyte-CSF (G-CSF), A4 cells proliferate with accompanying development to form cells which resemble mature, postmitotic neutrophils. The presence of high concentrations of IL-3 (100 units/ml) blocks the development of A4 cells even in the presence of GM-CSF plus G-CSF. A4 cell development to neutrophils is accompanied by major changes in the expression of protein kinase C (PKC) subspecies in these cells. The predominant subspecies present in multipotent A4 cells, as judged by direct chromatographic analysis, was the type III enzyme (alpha) subspecies, whereas in mature A4 cell neutrophils, the type II (beta I + beta II) enzymes were predominant. Phorbol esters added to immature A4 cells resulted in a proliferative response, but when added to postmitotic A4 cells resembling neutrophils they elicited a large increase in reactive oxygen intermediate production. This suggests that the type III (alpha) subspecies may mediate proliferative responses in stem cells, whilst the type II (beta I + beta II) enzymes are more important for the mature cell functions of postmitotic neutrophils. In cultures containing IL-3 (100 units/ml) both the type III, and also the type II subspecies were predominantly membrane-associated for prolonged periods (> 24 hours).(ABSTRACT TRUNCATED AT 250 WORDS)