Regulation of eukaryotic protein kinases by Pin1, a peptidyl-prolyl isomerase

Abstract

The peptidyl-prolyl isomerase Pin1 cooperates with proline-directed kinases and phosphatases to regulate multiple oncogenic pathways. Pin1 specifically recognizes phosphorylated Ser/Thr-Pro motifs in proteins and catalyzes their cis-trans isomerization. The Pin1-catalyzed conformational changes determine the stability, activity, and subcellular localization of numerous protein substrates. We conducted a survey of eukaryotic protein kinases that are regulated by Pin1 and whose Pin1 binding sites have been identified. Our analyses reveal that Pin1 target sites in kinases do not fall exclusively within the intrinsically disordered regions of these enzymes. Rather, they fall into three groups based on their location: (i) within the catalytic kinase domain, (ii) in the C-terminal kinase region, and (iii) in regulatory domains. Some of the kinases downregulated by Pin1 activity are tumor-suppressing, and all kinases upregulated by Pin1 activity are functionally pro-oncogenic. These findings further reinforce the rationale for developing Pin1-specific inhibitors as attractive pharmaceuticals for cancer therapy.

Keywords: Cis-trans isomerization; Eukaryotic protein kinase; Oncogenic pathway; Peptidyl-prolyl isomerase Pin1; Proline-directed protein kinase; Tumor suppressor.

Figure 1.. Pin1 catalyzes cis-trans isomerization of pSer/Thr-Pro protein motifs.

(A) pSer/Thr motifs in proteins are generated by proline-directed protein kinases. (B) Pin1 catalyzes the isomerization of the peptidyl-prolyl bonds in pSer/Thr-Pro motifs. Pin1-mediated conformational changes regulate the stability and activity of its substrates.

Figure 2.. Pin1 substrate binding sites and catalytic mechanism.

(A) Superposition of 3D structures of Pin1 complexed to the (i) peptide derived from the C-terminal domain of RNA polymerase II (CTD), PDB ID: 1F8A; and (ii) peptide derived from the Bromodomain-containing protein 4 (BRD4), PDB ID: 5UY9. The CTD peptide binds to the WW domain (green), and the BRD4 peptide binds to the PPIase domain (blue). Both substrate peptides are shown in gray, with the pSer-Pro motifs highlighted in brown. (B) Residues in the WW and PPIase domains that participate in interactions with substrates. (C) Structural representation of the “twisted amide” model of Pin1 catalysis. The transition state was modeled using the UCSF Chimera package (Pettersen et al., 2004) by replacing the methylene of the Pin1 transition-state analog inhibitor (PDB ID: 3NTP) with the carbonyl group. The rotation of the pSerPro amide bond upon isomerization is shown with green arrows. (D) Dynamic hydrogen bonding network in the active site of Pin1 is formed by Ser115, Cys113, His59, His157, and Thr152. The structure of the Pin1::BRD4 complex (PDB ID: 5UY9) was used to generate the structural representation. For clarity, only pSer-Pro fragments of the substrate peptides are displayed in (B) and (D). In (B-D), salt bridges and hydrogen bonds are shown with dashed red and black lines, respectively.

Figure 3.. Phosphorylation sites in Pin1 and regulatory outcomes.

The Pin1 phosphorylation sites are mapped onto the 3D structures of the (A) WW and (B) PPIase domains. The coordinates of the Pin1::CTD (PDB ID: 1F8A) and Pin1::Brd4 (PDB: ID 5UY9) complexes were used in (A) and (B), respectively; only the pSer-Pro motifs of the substrate peptides are displayed for clarity (brown). For visualization purposes, the phosphate groups were added to the Ser residues using the UCSF Chimera software package (Pettersen et al., 2004). The phosphorylation sites are labeled 1 through 5 according to the order mentioned in text. In (B), hydrogen bonds and the putative pSer71-Arg69 salt bridge are shown with dashed black and red lines, respectively. The catalytic loop (residues 63–80) is highlighted in pink. Residues 137–142 of the α4 helix in PPIase that contribute to the formation of the interdomain interface are colored gray. The kinase identity and regulatory outcomes of Pin1 phosphorylation are given next to the site labels (also see Table 1).

Figure 4.. Pin1 target sites in eukaryotic kinases.

The Pin1 target sites are grouped according to their location: (A) within the catalytic kinase domain, (B) in the C-terminal kinase region, and (C) in regulatory domains. The color-coding scheme is as follows: Pin1 (blue); catalytic kinase domain (gray); activation loop (black); C-terminal region (green); phosphorylated Pin1-interacting motifs (red). The reported outcomes of Pin1-mediated regulation are given for each kinase (also see Table 2).