Monomeric/dimeric forms of Fgf15/FGF19 show differential activity in hepatocyte proliferation and metabolic function.

Human Fibroblast Growth Factor 19 (FGF19) and mouse ortholog Fgf15 play similar roles in liver regeneration and metabolism via the activation of Fgfr4/b-klotho (Klb). Monomeric FGF19 and dimeric Fgf15 are both necessary for liver regeneration and proper bile acid (BA) metabolism. FGF19 elicits stronger effects than Fgf15 on glucose and fatty acid metabolism and only FGF19 induces hepatocellular carcinoma (HCC). However, inhibiting FGF19/FGFR4 signaling in HCC patients is associated with toxicity due to elevated BA levels. Here, we examine the structure/function relationship in Fgf15/FGF19 to better understand the molecular basis for their distinct functions. We demonstrate that FGF19 is a more effective activator of Fgfr4 and of downstream signaling (Erk, Plcg1) than Fgf15. Furthermore, we use site-directed mutagenesis to show that the presence or absence of an unpaired cysteine in Fgf15/19 modulates ligand structure and determines the ability of these molecules to induce hepatocyte proliferation, with monomers being more potent activators. Consistent with these findings, an engineered dimeric variant of FGF19 is less effective than wild-type FGF19 at inducing liver growth in cooperation with the Wnt-enhancer RSPO3. In contrast to effects on proliferation, monomeric and dimeric ligands equally inhibited the expression of Cyp7a1, the enzyme catalyzing the rate limiting step in BA production. Thus, structure and function of Fgf15/FGF19 are intricately linked, explaining why FGF19, but not Fgf15, induces liver tumorigenesis. Our data provide insight into FGF19/FGFR4 signaling and may inform strategies to target this pathway while limiting on-target toxicity due to dysregulation of BA production or induction of hepatocyte proliferation.

Pim kinase substrate identification and specificity.

The Pim family of Ser/Thr kinases has been implicated in the process of lymphomagenesis and cell survival. Known substrates of Pim kinases are few and poorly characterized. In this study we set out to identify novel Pim-2 substrates using the Kinase Substrate Tracking and Elucidation (KESTREL) approach. Two potential substrates, eukaryotic initiation factor 4B (eIF4B) and apoptosis inhibitor 5 (API-5), were identified from rat thymus extracts. Sequence comparison of the Pim-2 kinase phosphorylation sites of eIF4B and mouse BAD, the only other known Pim-2 substrate, revealed conserved amino acids preceding the phosphorylated serine residue. Stepwise replacement of the conserved residues produced a consensus sequence for Pim kinase recognition: RXRHXS. Pim-1 and Pim-2 catalyzed the phosphorylation of this recognition sequence 20-fold more efficiently than the original (K/R-K/R-R-K/R-L-S/T-a; a = small chain amino acid) Pim-1 phosphorylation site. The identification of the novel Pim kinase consensus sequence provides a more sensitive and versatile peptide based assay for screening modulators of Pim kinase activity.

Structural basis of constitutive activity and a unique nucleotide binding mode of human Pim-1 kinase.

Pim-1 kinase is a member of a distinct class of serine/threonine kinases consisting of Pim-1, Pim-2, and Pim-3. Pim kinases are highly homologous to one another and share a unique consensus hinge region sequence, ER-PXPX, with its two proline residues separated by a non-conserved residue, but they (Pim kinases) have <30% sequence identity with other kinases. Pim-1 has been implicated in both cytokine-induced signal transduction and the development of lymphoid malignancies. We have determined the crystal structures of apo Pim-1 kinase and its AMP-PNP (5'-adenylyl-beta,gamma-imidodiphosphate) complex to 2.1-angstroms resolutions. The structures reveal the following. 1) The kinase adopts a constitutively active conformation, and extensive hydrophobic and hydrogen bond interactions between the activation loop and the catalytic loop might be the structural basis for maintaining such a conformation. 2) The hinge region has a novel architecture and hydrogen-bonding pattern, which not only expand the ATP pocket but also serve to establish unambiguously the alignment of the Pim-1 hinge region with that of other kinases. 3) The binding mode of AMP-PNP to Pim-1 kinase is unique and does not involve a critical hinge region hydrogen bond interaction. Analysis of the reported Pim-1 kinase-domain structures leads to a hypothesis as to how Pim kinase activity might be regulated in vivo.

Expression, purification, crystallization and preliminary crystallographic analysis of human Pim-1 kinase.

Pim kinases, including Pim-1, Pim-2 and Pim-3, belong to a distinctive serine/threonine protein-kinase family. They are involved in cytokine-induced signal transduction and the development of lymphoid malignancies. Their kinase domains are highly homologous to one another, but share low sequence identity to other kinases. Specifically, there are two proline residues in the conserved hinge-region sequence ERPXPX separated by a residue that is non-conserved among Pim kinases. Full-length human Pim-1 kinase (1-313) was cloned and expressed in Escherichia coli as a GST-fusion protein and truncated to Pim-1 (14-313) by thrombin digestion during purification. The Pim-1 (14-313) protein was purified to high homogeneity and monodispersity. This protein preparation yielded small crystals in the initial screening and large crystals after optimization. The large crystals of apo Pim-1 enzyme diffracted to 2.1 A resolution and belong to space group P6(5), with unit-cell parameters a = b = 95.9, c = 80.0 A, beta = 120 degrees and one molecule per asymmetric unit.