Results

Three tryptic 124-v-Mos peptides include target sites for auto-phosphorylation
We have expressed 124-v-Mos with the baculovirus system in Sf9 insect cells and immunopurified 124-v-Mos using the anti-Mos N13 antiserum [19]. As a control, a Mos-unrelated protein, a synthetic kinase-inactive construct of PKC, PKCγK380R[27], was expressed in Sf9 cells. Mos kinase assays, completed in the presence of [γ-32P]ATP, were resolved using SDS-PAGE and the Coomassie blue staining of the protein gel showed visible amounts of immunopurified 124-v-Mos (fig. 1B, arrowhead). The corresponding autoradiograph in figure 1A demonstrates that 124-v-Mos is expressed as a constitutive active protein kinase indicated by its ability to auto-phosphorylate in vitro. Further, a parallel kinase reaction was used for phosphoamino acid analyses which confirmed that 124-v-Mos auto-phosphorylation occurred predominantly on serine residues (fig. 1C) and a two-dimensional resolution of a tryptic digest of auto-phosphorylated 124-v-Mos showed that three tryptic peptides include auto-phosphorylation target sites (fig. 1D), demonstrating that auto-phosphorylation occurs on multiple sites of the Mos protein [28].
Figure 1  Constitutive kinase activity of immunopurified 124-v-Mos from baculovirus expressing Sf9 insect cells. Auto-phosphorylation of immunopurified 124-v-Mos expressed in Sf9 cells is shown in B (Coomassie stained 10% SDS-PAGE) and A (corresponding autoradiograph). Parallel 124-v-Mos kinase assays were subjected to a two-dimensional phosphoamino acid analysis (C) or a tryptic digestion followed by a two-dimensional resolution (D). Arrowheads indicate the origin of sample application in (C,D) and the position of 124-v-Mos (A,B).

124-v-Mos phosphorylates vimentin but not tubulin in vitro
Initially, we tested the kinase activity of 124-v-Mos using previously identified Mos substrates. It has been shown that 124-v-Mos, derived from mos-transformed fibroblasts, phosphorylates vimentin in vitro [29] and as presented here in figure 2C, in vitro kinase assays using immunopurified 124-v-mos from Sf9 insect cells showed strong vimentin phosphorylation. In contrast, tubulin which has been shown to be phosphorylated in vivo and in vitro by Xenopus c-Mos [30] was not a substrate for 124-v-Mos in vitro (fig. 2A). We have tested tubulin purified from various organs (mouse brain, testis and spleen) either polymerised, unpolymerised or pretreated with phosphatases but in none of these states found tubulin to be phosphorylated by 124-v-Mos (data not shown).
Figure 2  124-v-Mos phosphorylates vimentin but not tubulin. In vitro 124-v-Mos kinase assays with either vimentin (C,D) or purified tubulin from brain (A,B) as substrates were electrophoresed using 10% SDS-PAGE and Coomassie stained (B,D), the corresponding autoradiographs are shown in (A,C). Immunoprecipitates of Sf9 cells expressing the kinase-inactive PKCγK380R were indicated as controls.

Demonstration of alpha and beta-casein phosphorylation by 124-v-Mos
In search of further substrates for the 124-v-Mos protein kinase we tested MBP; histone HI, H2AS, H3; protamine; protaminsulphate; purified PKC-α/-β II/γ and α- and β-casein. With the exception of α- and β-casein (fig. 3A) none of these substrates were phosphorylated by 124-v-Mos (data not shown). The possibility that factors other than 124-v-Mos in the immunoprecipitate might be responsible for the observed casein phosphorylation was eliminated by including a synthetic kinase-inactive construct of 124-v-Mos, 124-v-MosK121R[19], as a control in addition to the Mos-unreleated protein, PKC_K380R. A comparison of background phosphorylation on β-casein in the immunoprecipitates of both controls and 124-v-Mos specific phosphorylation showed that 124-v-Mos phosphorylates β-casein 7fold relative to background (fig. 3B). Critically, a tryptic digest of phosphorylated β-casein revealed that 124-v-Mos phosphorylates a specific tryptic peptide in β-casein which shows no background phosphorylation in either controls (fig. 3C, arrowhead) strongly supporting that 124-v-Mos is able to phosphorylate β-casein. Further, a two-dimensional phosphoamino acid analysis (fig. 3D) showed that 124-v-Mos phosphorylates α- and β-casein on serine and threonine residues at a ratio of 1:1.
Figure 3  124-v-Mos phosphorylates α- and β-casein in vitro. Mos kinase assays, in the presence of α- and β-casein, were resolved using 10% SDS-PAGE; the Coomassie stained protein gel shown in 3A, right panel and the corresponding autoradiograph on the left panel. Arrowheads indicate the position of 124-v-Mos, α- and β-casein and the antibody. Using two control immunoprecipitates of Sf9 cells expressing the synthetic kinase-inactive constructs, 124-v-MosK121R or PKCγK380R, Mos-specific β-casein phosphorylation was demonstrated in 3B and 3C: Mos kinase assays were blotted on nylon-membrane, the phospho-β-casein bands (B, arrowhead) excised and 32P-Cerenkov counts recorded (B). Alternatively, the excised phospho-β-casein bands were digested with trypsin and electrophoresed using 16% SDS-PAGE (C). The arrowhead in 3C indicates the tryptic β-casein peptide phosphorylated by wild-type 124-v-Mos only. Further, two-dimensional phosphoamino acid analyses of 124-v-Mos phosphorylated α-casein (D, left panel) and β-casein (D, right panel) were completed, the arrowheads indicating the origins of sample application.

The protein tyrosine phosphatase 1B is a novel in vitro substrate for 124-v-Mos
Protein tyrosine phosphatases constitute a diverse family of enzymes that can be divided into several subgroups, including receptor and non-receptor PTPs [31]. The non-transmembrane protein tyrosine phosphatase PTP-1B, a major intracellular PTP is widely expressed. PTP-1B has been demonstrated to be phosphorylated on multiple sites in a cell cycle specific manner whereby mitotic hyper-phosphorylation occurs, reflected by a protein mobility shift in SDS-PAGE analyses [32]. Using purified PTP-1B as a substrate, we show here that 124-v-Mos can phosphorylate PTP-1B in vitro (fig. 4A). We controlled this result by using immunoprecipitates from Sf9 cells expressing the synthetic kinase-inactive 124-v-Mos construct or purified PTP-1B alone in parallel kinase assays (fig. 4A). Other kinases such as PKC and CKII that phosphorylate PTP-1B in vitro are unable to induce a mobility shift of PTP-1B as observed in mitotic cells [32]. Likewise, as shown in figure 4B, a Mos-dependent phosphorylation did not result in a mobility shift of PTP-1B.
Figure 4  PTP-1B is a substrate for 124-v-Mos in vitro. In vitro Mos kinase assays, using purified PTP-1B as a substrate, were resolved using 10% SDS-PAGE and the autoradiograph is shown in 4A. Immunoprecipitates of Sf9 cells expressing the kinase-inactive 124-v-MosK121R variant or PTP-1B alone were included as controls (A,B). A parallel kinase assay was blotted on nylon-membrane and PTP-1B was detected (B) using the PTP-1B-specific antiserum FG6 [29], arrowheads indicate the position of 124-v-Mos and PTP-1B.