PASK activity is independent of activation loop phosphorylation. A, HEK293T cells were transfected with plasmids expressing the indicated variants of V5-tagged human PASK. At 24 h after transfection, cells were lysed, PASK was immunoprecipitated using anti-V5 antibody, and an in vitro kinase activity assay was performed using Ugp1 and [γ-³²P]ATP as substrates. Samples were separated by SDS-PAGE and subjected to autoradiography (top panel), anti-V5 Western blotting (middle panel) and Ponceau S staining (bottom panel). B, HEK293T cells were transfected with plasmids expressing either WT or T1161A mutant V5-tagged human PASK as indicated. At 24 h after transfection, cells were lysed, PASK was immunoprecipitated using anti-V5 antibody, and an in vitro kinase activity assay was performed using Ugp1 and [γ-³²P]ATP as substrates for the indicated time, followed by addition of SDS buffer to terminate the reaction. Samples were separated by SDS-PAGE and subjected to autoradiography (top and middle panels), and Ponceau S staining (bottom panel). C, HEK293T cells were transfected with plasmids expressing the indicated variants of V5-tagged human PASK. At 24 h after transfection, metabolic ³²P labeling was performed for 4.0 h. Following cell lysis, PASK was immunoprecipitated using anti-V5 antibody, and samples were separated by SDS-PAGE and subjected to autoradiography (top panel) and anti-V5 Western blotting (bottom panel). D, three separate experiments were quantified as described in C using Scion Image software. Mean ± S.D. (error bars) are shown.

Crystal structure of the kinase domain of PASK. A, secondary structure elements of PASK kinase domain in the presence of ADP are shown. The α-helices are depicted in red, β-strands in yellow, loops in green, and ADP and Mg²⁺ ions in blue. The strands and helices are named according to the conventional nomenclature for PKA. The N and C termini of the kinase structure are indicated as N and C, respectively. B, PASK three-dimensional structure is rotated 180° along the vertical axis to show the positions of βH1 and βH2 helices (marked). See “Results” for details.

Unusual N-terminal lobe structure in PASK is shared by PIM1. A, comparison of the βH1, βH2, and αC elements of the PASK and PIM1 N-terminal lobes is shown. The positions and orientations of the tryptophan (Trp¹⁰⁴⁰ in PASK) and phenylalanine (Phe¹⁰⁷⁷ in PASK) residues are shown in each structure. Side chains are colored by element. B, orientation and interactions of important and conserved ATP binding pocket residues are normal despite the insertion of the βH1/βH2 β-hairpin in PASK. Glu¹⁰⁵² from the αC helix forms an ionic interaction with Lys¹⁰²⁸ and ADP (black) as typical of most active protein kinase structures.

Interaction of the PASK N-terminal lobe with the C-terminal lobe in the absence of Thr¹¹⁶¹ phosphorylation. A, comparison of the interaction between the N- and C-terminal lobes in an activation loop phosphorylation-dependent RD kinase (PDK1, left panel, PDB code 1H1W) or activation loop phosphorylation-independent RD kinase (CHK1, right panel, PDB code 1IA8). B, stabilization of the interaction between the N- and C-terminal lobes of PASK in the absence of activation loop phosphorylation by an Arg¹⁰⁵⁸-Tyr¹¹⁵² hydrogen bond. Asp¹⁰⁴³ from βH1 provides an additional anchor point for the Arg¹⁰⁵⁸ side chain conformation. C, loss of the interaction between the PASK N- and C-terminal lobes resulting in decreased in vitro kinase activity. WT PASK and the indicated point mutants were analyzed as described in Fig. 1A.

Alanine 1151 enables PASK catalytic activity in the absence of activation loop phosphorylation. A, sequence alignment of PASK with several protein kinases is shown. The basic residue at the DFG+3 position is indicated by the inverted triangle. This basic residue is typically found in kinases regulated by activation loop phosphorylation (dependent), but not in activation loop phosphorylation-independent kinases (independent). B, A1151K substitution decreases PASK activity, which is partially rescued by a phosphomimetic T1161E substitution in the activation loop. WT PASK and the indicated point mutants were analyzed as described for Fig. 1A.

Stabilization of catalytic core residues in PASK in the absence of activation loop phosphorylation. A, polar interactions of the catalytic loop and Mg²⁺-binding loop with ADP and αC helix residues. ADP and Mg²⁺ are indicated. C-α atoms of Gly¹¹⁴⁸ and Ser¹¹⁴⁹ that form hydrogen bonds with the Asp¹¹⁴⁶ and Arg¹¹²⁷ side chains, respectively, are shown. For simplicity, C-α of other amino acids not involved in hydrogen bond formation are not shown. B, structural comparison of the catalytic core of PASK, PIM1, and glycogen synthase kinase 3β (PDB code 1I09). Tyrosine analogous to Tyr¹¹⁷⁹ in all three kinases hydrogen bonds with the arginine from the catalytic loop, which in turn hydrogen bonds with the backbone of the serine in the Mg²⁺ binding loop. Tyr¹¹⁷⁹ of PASK and its structural homolog in PIM1 also forms a hydrogen bond with the main chain oxygen of the activation loop residue. See “Results” for details. C, active structures of AKT (PDB code 1O6K) and ERK1 (PDB code 2ERK) shown to illustrate the polar contacts involving the tyrosine, activation loop, and RD pocket. D, inactive structures of AKT (PDB code 1MRY) and ERK1 (PDB code 1ERK) showing a lack of hydrogen bonding between the tyrosine and RD pocket. In the AKT structure, the Mg²⁺-binding loop and activation loop are not ordered. Tyr³²⁷ of AKT interacts with Asn³²⁵ via a solvent molecule in the inactive conformation.

Structural basis for the substrate specificity of the PASK kinase domain. A, full-length human PASK was used to phosphorylate a 198-member combinatorial peptide library. Peptides had the generic sequence YAXXXXXS/TXXXXAGKK (biotin), with the indicated residue (columns) fixed at the indicated position (row) relative to the central phosphoacceptor site. B, stereo view of the PASK kinase domain (blue) is superimposed on the PIM1 structure (red) bound to PIMTIDE substrate peptide (green; sequence KRRRHPS).