Rasar M et al. (2006), Paxillin regulates steroid-triggered meiotic re...

XB-ART-34724

J Biol Chem 2006 Dec 22;28151:39455-64. doi: 10.1074/jbc.M608959200.

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Paxillin regulates steroid-triggered meiotic resumption in oocytes by enhancing an all-or-none positive feedback kinase loop.

Rasar M , DeFranco DB , Hammes SR .

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Oocyte maturation is triggered by steroids in a transcription-independent fashion that involves an unusual positive feedback loop whereby MOS (a germ cell-specific Raf) activates MEK1, which in turn activates ERK2. ERK2 then acts back on MOS to enhance its expression and amplify the kinase signaling cascade. To date, little is known regarding other factors that regulate this powerful positive feedback kinase cascade. Here we present the scaffold molecule Paxillin as a newly recognized essential regulator of meiosis in Xenopus laevis oocytes. Reduction of Paxillin expression using RNA interference or antisense oligonucleotides completely abrogates steroid-triggered meiotic resumption. Detailed signaling studies reveal that Paxillin is acting early in the kinase cascade, because it is required for accumulation of MOS protein and complete activation of downstream kinase signaling in response to steroids. Surprisingly, full Paxillin activity also requires serine phosphorylation by a kinase downstream of MOS and MEK1, possibly ERK2. Together, these data suggest that Paxillin is an important regulator of the positive feedback effects of MEK/ERK signaling on MOS protein expression. These experiments reveal a novel and critical function for Paxillin in meiosis and support the notion that Paxillin may be a general modulator of mitogen-activated protein kinase signaling.

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Species referenced: Xenopus laevis
Genes referenced: camp ccnb1.2 cdk1 cdk20 ins map2k1 mapk1 mos pxn
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	FIGURE 1. Schematic of Paxillin functional domains. XePaxillin has 80% homology to mammalian isoforms of Paxillin. The N-terminal half has five LD motifs and the C-terminal half has four LIM domains. Between the first and second LD motif are three conserved phosphorylation sites. The first two regions, 31-YSFP-34 and 99-YSFP-102, are SH2 binding sites at which tyrosine phosphorylation has been reported. The third region, 107-SAEPSP-112, contains two serine residues that are conserved Paxillin phosphorylation sites.
	FIGURE 2. Paxillin is required for steroid-induced oocyte maturation. A, oocytes were injected with HAtagged XePaxillin cRNA (XePax) to increase XePaxillin expression, double-stranded XePaxillin cRNA (ds XePax) to decrease XePaxillin expression, or 10 mM Hepes (Mock). After 48 h, oocytes were incubated with the indicated concentrations of testosterone and maturation (germinal vesicle breakdown) was scored after 16 h. B, oocytes were injected with either sense or antisense oligonucleotides without and with HA-XePaxillin cRNA and then treated as in A. Rescued oocytes contained HA-XePaxillin as measured by Western blot using an anti-HA antibody (inset). Results of dsRNA and antisense knockdown studies are representative of at least five experiments each. C, immunohistochemistry was performed on paraffin-embedded sections of mock, XePaxillin dsRNA, XePaxillin sense oligonucleotide, or XePaxillin antisense oligonucleotide injected oocytes. Sections were incubated with equal concentrations of rabbit anti-Paxillin antisera or the corresponding preimmune serum. These are representative photos of multiple slides. D, the anti-Paxillin, but not the corresponding pre-immune, serum recognizes the C-terminal half of XePaxillin when overexpressed in oocytes. Oocytes were injected with cRNAs encoding the indicated proteins and Western blots performed after 48 h. E, XePaxillin mRNA is present in sense-injected oocytes but disappears in oocytes injected with XePaxillin antisense oligonucleotides, as detected by Northern blot (upper panel). Northern blots correspond to the mRNA prepared for Fig. 2D. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels were unchanged (lower panel).
	FIGURE 3. Knockdown of Paxillin blocks accumulation of MOS and activation of p42 ERK. A, oocytes were injected with HA-tagged XePaxillin (Pax), XePaxillin dsRNA (dsPax), or Hepes (Mock). After 48 h, oocytes were incubated with 150 nM testosterone, 50 g/ml insulin, or 0.1% ethanol. Oocytes were permeabilized at 4 and 8 h and immunoblottedfor HA-Pax, MOS (after 8 h), or phospho-p42 ERK (p42-PO4) (after 4 h). Phospho-p42 ERK blots were stripped and re-probed for total p42 ERK. B, oocytes injected with either sense or antisense Paxillin oligonucleotides were stimulated with 300 nM testosterone, and lysates were probed as in panel A. C and D, oocytes were injected with the indicated reagents as described above. Oocytes were then injected with 10 ng of Mos 3UTR polyadenylation reporter RNA after 40 h and stimulated with 300 nM testosterone or ethanol. RNA was extracted at the indicated times, and equal amounts of RNA were loaded per lane. Polyadenylation of the 3UTR reporter was analyzed by Northern blot using radiolabeled Mos 3UTR DNA as the probe. Slower migration of RNA represents increased polyadenylation. E and F, MOS rescues the inhibition of maturation from decreased Paxillin expression. Oocytes were injected with 5 ng of Mos cRNA or 10 mM Hepes (Mock) 40 h after injection of oocytes with sense or antisense Paxillin oligonucleotides. Oocytes were permeabilized after 18 h and lysates probed for MOS and phospho-p42 ERK (E). The lower panel represents a stripped phospho-p42 ERK blot probed for total p42 ERK. The percentage of spontaneous maturation induced by injection of Mos cRNA (MOS) after 18 h was also measured (F). All data represent one of at least three experiments with essentially identical results. Twenty oocytes per point were used for each data set, and the experiments for A and C were performed on the same set of injected oocytes at the same time with the same reagents, as were the experiments in B, D, E, and F.
	FIGURE 4. Paxillin is phosphorylated late during maturation in a MEK-dependent fashion. A, oocytes were stimulated with 1 M testosterone (to ensure maximum signaling) 40 h after injection of oocytes with HA-Paxillin cRNA. Oocytes were permeabilized at the indicated times, and 30 l of the sample was treated with calf intestinal alkaline phosphatase. Equal amounts of lysate were analyzed by SDS-PAGE followed by Western blot with and anti-HA antibody. The higher mobility bands represent phosphorylated Paxillin. B, oocytes were injected with either HA-tagged cRNA encoding full-length (FL) Paxillin, the N-terminal half of Paxillin (N-Paxillin, residues 1–304), or the C-terminal half of Paxillin (C-Paxillin, residues 291–539). 40 h post-injection, oocytes were stimulated with 1M testosterone, permeabilized at the indicated times, and immunoblotted with HA antibody. C, lysatesfrom Awere probed with MOS antibody.Dand E, 40 h post-injection, HA-Paxillin-expressing oocytes were pretreated with either Me2SO or 50 M PD98059 for 2 h before stimulation with 1 M testosterone (D) or injection with Mos cRNA (E). Either Me2SO or PD98059 was maintained in the media throughout the experiment. Oocytes were permeabilized at the indicated times and lysates probed for HA, MOS, and phospho-p42 ERK2. F, COS-7 cells were transfected with a total of 1g of DNA that consisted of pcDNA3.1, cDNAs encoding either HA-Paxillin (0.5 g) or HA-MOS (0.5 g), or both HA-Paxillin and HA-MOS (0.5 g each). After 18 h, cells were starved for 24 h, and then treated with either 50 M PD98059 or Me2SO vehicle for 2 h. Cells were permeabilized, and equal amounts of lysate were probed for HA and phospho-ERKs. Phospho-ERK blots were stripped and re-probed for total ERK. Each experiment was reproduced at least three times with essentially identical results. Oocytes in A–E were from the same batch and treated as indicated at the same time.
	FIGURE 5. Paxillin phosphorylation and function depends on serine residues 107 and 111. A, oocytes were injected with HA-tagged Paxillin cRNA that encoded either wild-type Paxillin (WT-Pax), singly mutated Paxillin (S107A or S111A), or doubly mutated Paxillin (S107/111A). 40 h post-injection, oocytes were stimulated with or without 1 M testosterone for 16 h, and lysates were probed with HA antibody. B, oocytes depleted of Paxillin by antisense injection (AS) were co-injected with either wild-type Paxillin (WTPax) or the double serine to alanine mutant Paxillin (S107/111A-Pax). Oocytes were then incubated with increasing doses of testosterone. Twenty oocytes were used for each data point. C, lysates from the oocytes treated with 250 nM testosterone fro 16 h in B were analyzed for MOS expression and p42 ERK activation by Western blot. Phospho-p42 ERK blots were stripped and reprobed for total p42 ERK. D, equivalent expression of wild-type and S107A/ S111A Paxillin in the oocytes from B was verified by HA immunoblot. Each study was performed at least three times with essentially identical results.
	FIGURE 6. p42 ERK phosphorylates Paxillin in vitro. A, GST fusion proteins of full-length wild-type Paxillin (WT-Pax), S107A/S111A Paxillin (S107/111APax), or GST alone were incubated with either inactive or active ERK2 in the presence of 10 Ci of [32P]ATP. No phosphorylation of GST alone was observed (data not shown). B, Western blots show equal, if not greater, loading of S107A/S111A Paxillin compared with WT-Paxillin.
	FIGURE 7. Proposed model describing Paxillin function during oocyte maturation. Testosterone stimulation of oocytes via the classic androgen receptor triggers a decrease in intracellular cAMP. This drop in cAMP leads to increased polyadenylation of Mos mRNA, resulting in a small increase in MOS protein expression. MOS then activates MEK1, which in turn activates ERK2. Activated ERK2, and possibly other intracellular kinases, then phosphorylate Paxillin, which acts to further enhance MOS protein expression by either increasing MOS protein translation, decreasing MOS degradation, or both. This powerful positive feedback loop eventually leads to activation of the maturation promoting factor (MPF, or cyclin-dependent kinase 1 (CDK1), and cyclin B), resulting in meiotic resumption.