p90^rsk kinase activity is present in unfertilized Xenopus eggs. (A) Assays for p90^rsk activity were carried out in extracts from unfertilized Xenopus eggs by using an in-gel kinase assay as described in Materials and Methods. In the absence of an exogenous substrate (Crosstide), several phosphorylated proteins were seen in the egg protein extract (lane 1). In the presence of Crosstide, a 90-kDa kinase activity is increased, indicating specific phosphorylation of the exogenous substrate (lane 2). (B) The possibility that the 90-kDa kinase activity is p90^rsk was tested by using FPLC Mono-Q fractionation followed by Crosstide kinase assays and Western blot analysis. A peak of Crosstide kinase activity was eluted at approximately 0.1 M NaCl in fractions 24 to 36. Western blot analysis confirmed the presence of p90^rsk kinase in these fractions (inset). The majority of Crosstide kinase activity was present in the flowthrough (data not shown), including fractions 1 to 10 as the shoulder of this activity; however, p90^rsk was not present in the flowthrough fractions. (C) In-gel kinase assays performed without an exogenous substrate reveal a 90-kDa kinase activity present in unfertilized egg and in 2-, 32-, and 128-cell embryo extracts (lanes 1 to 4, top gel). Immunodepletion of p90^rsk kinase from these protein extracts with an anti-p90^rsk polyclonal antibody prior to the in-gel kinase assays results in the removal of the 90-kDa band (bottom gel), demonstrating that the 90-kDa kinase activity band is p90^rsk.

Overexpression of p90^rsk leads to increased steady-state levels of endogenous β-catenin. (A) XS6KIIA mRNA (total of 30 or 3 ng) encoding Xenopus p90^rsk kinase was microinjected into the marginal zone of both dorsal or ventral blastomeres at the four-cell stage (4DMZ and 4VMZ, respectively). Total β-catenin and p90^rsk steady-state levels were determined by Western blot analysis. Small elevations in p90^rsk protein levels are sufficient to increase the steady-state levels of endogenous β-catenin (compare control lane 4 with XS6KIIA-injected lanes 5 and 6). α-Spectrin Western blot analyses were performed with the same nitrocellulose filter to control for protein extract quality and gel loading. After normalization to α-spectrin levels, microinjection of XS6KIIA mRNA (3 or 30 ng) into the dorsal or ventral marginal zones of Xenopus embryos leads to a statistically significant 215% increase in total endogenous β-catenin steady-state levels relative to untreated controls (n = 7, P < 0.01 by Student’s t test, SE, 24; compare control lanes 1 and 4 to XS6KIIA-injected lanes 2 and 3 or 5 and 6). Experiments with either 30 or 3 ng of injected XS6KIIA RNA yielded identical results. (B) N/C XS6KIIA mRNA (2.5 and 3.5 ng) encoding a mutant, putative kinase-dead N/C p90^rsk with conserved ATP-binding domain lysines 94 and 445 changed to arginines was microinjected into the marginal zone of both dorsal or ventral blastomeres at the four-cell stage (ventral microinjection experiment shown). Overexpression of N/C p90^rsk does not increase total steady-state levels of endogenous β-catenin (compare control lane 1 to N/C XS6KIIA-injected lanes 2 and 3), with average β-catenin levels of 92% (n = 4) relative to controls, after normalization to α-spectrin signal on the same Western blot.

Overexpression of p90^rsk increases the amount of membrane-associated β-catenin. (A) XS6KIIA mRNA (1.5 ng) encoding p90^rsk was microinjected into the marginal zone of both blastomeres of two-cell embryos. β-Catenin–myc mRNA (0.1 ng) was microinjected into the marginal zone of a single blastomere at the four-cell stage. Green fluorescent protein (GFP) (1.5 ng) and β-galactosidase (1.5 ng) mRNAs were used to control for nonspecific effects and to maintain a constant amount of microinjected mRNA. To determine whether the overexpression of p90^rsk alters the amount of β-catenin associated with cadherins, embryonic protein was extracted in RIPA buffer in the presence of 1% Nonidet P-40 0.5% sodium deoxycholate, and 0.1% SDS to extract membrane-associated β-catenin. ConA-Sepharose beads were used to obtain a cadherin-enriched fraction, whose endogenous and exogenous β-catenin content was analyzed by anti-β-catenin (top gel) and anti-c-myc Western blotting (bottom gel), respectively. Overexpression of p90^rsk leads to an accumulation of exogenous β-catenin–myc (compare lanes 2 and 3 and lanes 5 and 6, bottom gel). This effect also was observed for endogenous β-catenin but only after λ-phosphatase (λ PPase) treatment (compare lanes 5 and 6, top gel). This suggests that the N-terminal epitope detected by the polyclonal anti-β-catenin antibody used to detect endogenous β-catenin is partially masked by phosphorylation after p90^rsk overexpression (compare lane 3 to lanes 1 and 2, top gel). This masking effect is not observed when exogenous β-catenin is detected by using the C-terminal anti-c-myc antibody (bottom gel). Only trace amounts of β-catenin and β-catenin–myc were detectable by enhanced chemiluminescence in the supernatant fractions of these experiments (data not shown). (B) Overexpression of p90^rsk does not lead to an increase in the steady-state levels of free β-catenin. Cytosolic protein from embryos treated as described above was extracted in buffer H in the absence of detergents. Endogenous and exogenous β-catenin were detected by Western blotting with a polyclonal anti-β-catenin antibody and a monoclonal anti-c-myc antibody, respectively. Overexpression of p90^rsk did not result in increased steady-state levels of endogenous, free (compare XS6KIIA-injected lane 3 to control lanes 1 and 2, top gel) or overexpressed, free myc-tagged β-catenin (compare XS6KIIA-injected lane 3 to control lane 2, bottom gel). These experiments were repeated three times with identical results in each case. (C) To simultaneously visualize β-catenin present in the cadherin-enriched ConA-Sepharose pellet and supernatant fractions, we repeated our ConA enrichment experiments (A) after microinjection of in vitro-translated [³⁵S]β-catenin, followed by microinjection of RNAs. The λ-phosphatase treatment was omitted from these experiments. As expected, microinjection of XS6KIIA (3 ng) but not prolactin (3 ng) RNA resulted in increased levels of [³⁵S]β-catenin associated with the ConA pellet (compare lanes 2 and 3) and not the supernatant fractions (compare lanes 5 and 6), as monitored by phosphorImager analysis.

FGF signaling inhibits endogenous GSK-3 activity. eFGF (10 ng) or eFGF (10 ng) with XS6KIIA (10 ng) mRNA was injected into the marginal zone of two-cell Xenopus embryos. At embryonic stage 8, proteins were extracted from 50 embryos per sample in modified buffer H with a final Triton X-100 concentration of 0.2%. The samples were then pooled to generate approximately 12 mg of total protein from 450 uninjected or FGF-overexpressing embryos and subjected to FPLC Mono-Q fractionation. Fractions 1 to 5 and 6 to 15 represent the flowthrough and wash fractions, respectively. GSK-3 activity was assayed by incubating fraction aliquots with kinase reaction mix, [γ-³²P]ATP, and p9Creb peptide substrate for 15 min at 30°C (see Materials and Methods). The kinase reaction mixtures were then spotted on p81 paper and counted for radioactivity. ³²P incorporation into the p9Creb peptide substrate is presented. On average, peak uninjected control kinase activity resulted in a transfer of 1.2 pmol of [³²P]phosphate from [γ-³²P]ATP to p9Creb peptide (n = 4; 1.5 pmol or 7640 cpm in the experiment shown). This peak correlated with highest level of endogenous GSK-3 protein found in the fractions by Western blot analysis. Overexpression of FGF reduced peak activity to 53% of control levels (4,020 cpm). Again, kinase activity correlated with GSK-3 protein levels (peaks in fraction 9) as determined by Western blotting (top blot). FGF overexpression resulted in the shift of the peak to fraction 11 (third blot from the top). FGF treatment increased GSK-3 serine 9 phosphorylation, as assayed by Western blotting with a monoclonal anti-phosphoserine 9 GSK-3 antibody (compare control fractions [second blot from top] with FGF-treated fractions [bottom blot]). This strongly suggests that FGF signaling inhibits GSK-3 by inducing the phosphorylation of this regulatory residue. The inhibition of GSK-3 activity by microinjection of eFGF and XS6KIIA mRNA together (average 52% of control levels, n = 3 experiments) was almost identical to that obtained by overexpression of FGF alone (average 48% of control levels, n = 2). The GSK-3 inhibition shown in this experiment is representative of that observed in all other experiments performed (n = 5). Kinase activity is reported in counts per minute, since equal amounts of total protein were present in the corresponding uninjected and FGF-treated fractions, as determined by the Bradford assay.

p90^rsk kinase is present in Xenopus embryos. (A) Microdissection of 32-cell embryos into animal and vegetal or into dorsal and ventral halves followed by in-gel kinase assays reveals that p90^rsk activity is present almost exclusively in the animal hemisphere of Xenopus embryos (compare lanes 1 and 2). The dorsal and ventral halves appear to possess approximately equivalent amounts of p90^rsk activity (lanes 3 and 4, respectively). (B) Western blot analysis demonstrates that p90^rsk protein is present in unfertilized Xenopus eggs and in Xenopus embryos at various early stages of development (lanes 1 to 5). To control for protein extract quality and gel loading, α-spectrin Western blots were performed with the same nitrocellulose filter (top of gel). In unfertilized egg extracts, a portion of the p90^rsk displays an electrophoretic mobility shift, consistent with p90^rsk hyperphosphorylation and/or activation (lane 1).

Overexpression of p90^rsk leads to increased steady-state levels of exogenous β-catenin–myc and this effect is antagonized by N/C p90^rsk. β-Catenin–myc mRNA (0.5 ng) was microinjected into the marginal zone of both blastomeres of two-cell Xenopus embryos in combination with XS6KIIA (3 ng) and/or N/C XS6KIIA (1.5 or 3 ng) mRNA, which encode p90^rsk and putative kinase-dead N/C p90^rsk, respectively. Control β-galactosidase (β-gal) mRNA was added to the mRNA mix to keep the total amount of mRNA microinjected constant (11 ng). Microinjection of XS6KIIA (lane 4) but not N/C XS6KIIA (lane 3) mRNA leads to the increased accumulation of β-catenin–myc (compared to control lane 2 [Fig. 3 and Table 1]), but not α-spectrin detected on the same nitrocellulose blot. Microinjection of increasing amounts of N/C XS6KIIA mRNA along with XS6KIIA mRNA antagonizes this effect (compare lane 4 with decreasing signals in lane 5 and then lane 6). This experiment was repeated three times with comparable results in each case.

Overexpression of p90^rsk and eFGF leads to increased p90^rsk kinase activity in Xenopus embryos. (A) XS6KIIA (10 ng), N/C XS6KIIA (13 ng), eFGF (12 ng), and dnFGFR (6 ng) mRNAs were microinjected alone or in combination into the marginal zone of both blastomeres of two-cell Xenopus embryos. Proteins were extracted at stage 8 from 10 to 15 embryos per sample in buffer H in the presence of 1% Triton X-100. p90^rsk was immunoprecipitated from these extracts with polyclonal anti-p90^rsk antibodies and then incubated with kinase reaction mix, [γ-³²P]ATP, and S6 peptide substrate. A 25-μl sample of the kinase reaction mixture was spotted on p81 paper after 15 min of incubation at 30°C. ³²P incorporation into the S6 peptide substrate is presented. On average, uninjected control p90^rsk kinase reactions resulted in a transfer of 106 fmol of [³²P]phosphate from [γ-³²P]ATP to the S6 peptide substrate (n = 9). For each experiment, all samples were normalized to control p90^rsk kinase activity levels, with controls set at 100% kinase activity. Overexpression of p90^rsk by microinjection of XS6KIIA mRNA increased p90^rsk kinase activity in a statistically significant manner to 414% relative to controls (P < 0.01 by Student’s t test, n = 8). Overexpression of the mutant, putative kinase-dead N/C p90^rsk by microinjecting N/C XS6KIIA mRNA caused a small increase in kinase activity (151%; P < 0.05 by Student’s t test, n = 3). Despite this effect, coinjection of N/C XS6KIIA mRNA and XS6KIIA mRNA resulted in an inhibition in p90^rsk kinase activity from 414 to 251% (P < 0.02 by Student’s t test, n = 4), although this level of kinase activity was still significantly greater than in the controls (P < 0.05 by Student’s t test, n = 4). Microinjection of eFGF mRNA doubled p90^rsk kinase activity (200%, n = 2), and overexpression of p90^rsk and FGF by coinjecting XS6KIIA and eFGF mRNA led to a synergistic, statistically significant stimulation of p90^rsk kinase activity to 17,717% above control levels (P < 0.005 by Student’s t test, n = 4). This treatment is also significantly greater than the 414% p90^rsk activity level obtained by overexpressing p90^rsk alone (P < 0.002 by Student’s t test, n = 4). Finally, coinjection of XS6KIIA and dnFGFR mRNA to overexpress p90^rsk along with a dnFGFR which blocks endogenous FGF signaling led to a 166% level of p90^rsk kinase activity relative to controls (P < 0.05 by Student’s t test, n = 4). This level of kinase activity is lower than that obtained from overexpressing p90^rsk alone (compare 414% to 166%). Asterisks indicate that the difference in kinase activity relative to controls is statistically significant (P < 0.05 for a single asterisk; P < 0.005 for a double asterisk). Squares indicate that the difference in kinase activity relative to XS6KIIA mRNA treatment is statistically significant (P < 0.02 for a single square; P < 0.002 for a double square). Error bars represent SE. (B) Microinjection of eFGF (4 ng), but not prolactin (4 ng), mRNA also stimulates p90^rsk kinase activity in the in-gel kinase assay, as evident by ³²P incorporation being higher in the p90^rsk band in the eFGF-treated samples (lane 3) relative to control samples (lanes 1 and 2) in the presence of Crosstide peptide substrate (n = 3 experiments).