Critical Role of WW Domain Phosphorylation in Regulating Phosphoserine Binding Activity and Pin1 Function

Abstract

Phosphoserine-binding modules help determine the specificity of signal transduction events. One such module, the group IV WW domain, plays an essential role in targeting the phosphorylation-specific prolyl isomerase Pin1 to its substrates. These modules require Ser/Thr phosphorylation of their ligands for binding activity. However, phosphorylation of these modules and its functional significance have not been described, nor is it known whether the function of Pin1 is regulated. Here Compelling evidence indicates that phosphorylation on Ser or Thr residues (pSer/Thr) also promotes the formation of proteinprotein (1-3). Small pSer/Thr-binding protein modules reminiscent of SH2 1 domains involved in recognition of phosphorylated Tyr have been recently described, which include group IV WW domains and FHA domains (2-7). However, the role of phosphorylation of these modules themselves has not been described. The best characterized pSer/Thr-binding WW domain is one in the peptidyl-prolyl isomerase (PPIase) Pin1 (4, 7). Pin1 specifically isomerizes the pSer/Thr-Pro bond (8 -10). Pin1 is essential for mitotic progression (11-13) and is required for the DNA replication checkpoint (14). Pin1 substrates are a defined subset of phosphorylated proteins, including many MPM-2 antigens (8,13,(15)(16)(17)(18)(19)(20). Pin1-catalyzed prolyl isomerization regulates the conformation and function of phosphoproteins (15, 17) and also facilitates dephosphorylation (12,15,17). Thus, Pin1-dependent peptide bond isomerization is a critical post-phosphorylation regulatory mechanism (12). However, it is likely that Pin1 is regulated by a post-translational modification.The first and essential step in Pin1-dependent regulation of targets is the substrate binding activity mediated by its WW domain (10 MATERIALS AND METHODS Interaction of Pin1 Mutants and MPM-2 Antigens orPhosphopeptides-GST fusion proteins were expressed and purified, and their ability to bind mitotic phosphoproteins was assayed as described (8,15). Peptide binding was measured as described (4).Localization Analysis of GFP-Pin1 and Its Mutant Proteins-Pin1 or its mutants were subcloned into pEGFP-C1 or pERFP-N1 (CLON-TECH), respectively. HeLa cells were transfected with pGFP-or pRFPPin1 or its mutants using Superfect (Qiagen). After 6 -24 h, cells were fixed and examined under a fluorescence microscope (Nikon).In Vivo and in Vitro Phosphorylation of Pin1-To detect Pin1 phosphorylation in vivo, HeLa cells were labeled with [ 32 P]orthophosphate, and Pin1 was immunoprecipitated with anti-Pin1 or anti-HA antibodies, followed by SDS-PAGE and autoradiography (4). 32 P-labeled Pin1 proteins were extracted and digested with trypsin followed by twodimensional chromatography (23). In vitro phosphorylation of Pin1 and its mutants were carried out as described (4). To detect the mobility shift of Pin1 during the cell cycle, HeLa cells were arrested at the G 1 /S boundary and released to enter the cell cycle as described (15). Cell lysates were subjected to low bis-SDS-PAGE, followed ...

C-terminal domain small phosphatase 1 and MAP kinase reciprocally control REST stability and neuronal differentiation

“…The 861 SPP 864 SP site in REST comprises a motif for peptidylprolyl cis/trans isomerase (Pin1) binding (14). We hypothesized that Pin1 interacts with REST at this site.…”

“…Pin1 binding to prolines requires phosphorylation of an adjacent serine or threonine (14). Because both serines 861 and 864 are highly predicted target sites for the prolinedirected kinases ERK1 and 2, we hypothesized that ERK and its upstream activators, EGF and the small GTPase Harvey rat sarcoma viral oncogene homolog, H-Ras, would promote phosphorylation at these sites in REST.…”

Semidominant Mutations in Reduced Epidermal Fluorescence 4 Reduce Phenylpropanoid Content in Arabidopsis

“…Although no informative largescale domains could be recognized within REF4, many short peptide motifs were identified, two of which were completely conserved across REF4 and its homologs: a class IV WW protein-interaction domain (DWPSPA) (Sudol and Hunter 2000), which is a submotif within a proline-directed serine kinase phosphorylation site (DWPSPAA) (Lu et al 2002). Interestingly the substitution of the conserved P919 in the suppressor mutant abolishes both of these motifs, consistent with the hypothesis that they may be important for REF4 function.…”

Pin1 overexpression is associated with poor differentiation and survival in oral squamous cell carcinoma

“…Pin1 overexpression can confer transforming properties on mammary epithelial cells that is associated with tumorigenesis (34). In addition, anti-sense Pin1 or dominantnegative Pin1 causes Pin1 depletion and apoptosis in cancer cells that indicates inhibition of Pin1 activity might kill cancer cells (33,34). Furthermore, the strong relationship between the Pin1 level and the clinical outcome of oral (Fig.…”

“…Non-tumor or oral squamous cell carcinoma tissues were pulverized freshly in liquid nitrogen and the lysates were directly subjected to immunoblotting analysis with antiPin1 antibodies, followed by semi-quantification of protein levels using NIH image, as described (12,33). By using antiPin1 antibodies and immunoblotting, all normal human oral tissues and their tumor parts showed immuno-reactivity.…”