PMC

Human, fly, and yeast PASK genes exhibit three regions of conservation. (A) Schematic diagram of the domain architecture of PASK proteins. (B–D) Alignment of the three conserved regions of the human, fly, and two yeast PASK proteins. Residues identical or similar in at least three of four PASK proteins are shaded in black and gray, respectively. (B) The N-terminal PAS domain (PAS-A). (C) The C-terminal PAS domain (PAS-B). (D) The serine/threonine kinase domain. Activation loop residues S1149, T1161, and T1165 are marked with asterisks.

hPASK protein is predominantly cytosolic. (A) HeLa cells were fractionated into 10⁵ × g soluble (cytosol) and pellet fractions. The 10⁵ × g pellet was extracted with 0.4 M NaCl (nucleus). Cytosolic and nuclear extracts were analyzed by Western blotting by using a polyclonal α-PASK antibody (U2501). (B) Immunostaining of HEK293 cells transfected with an expression vector encoding a V5 epitope-tagged version of hPASK. Cells were fixed and stained by using α-V5 monoclonal antibody and visualized by using FITC-tagged secondary antibody.

PAS-A specifically inhibits enzymatic activity of hPASKΔN. (A) Native hPASK and hPASKΔN were incubated with AMARA peptide and [γ-³²P]ATP in the presence or absence of various amounts of hPASK PAS-A. Reactions were quenched after 5 min, and incorporation of ³²P into peptide substrate was determined by liquid scintillation counting of purified peptide. Incorporation of ³²P into peptide per minute is plotted with error bars indicating SD. (B) hPASKΔN was incubated with AMARA peptide and [γ-³²P]ATP in the presence of hPASK PAS-A, NPAS2 PAS-A, and EPAS1 PAS-B at the concentrations indicated. Reactions were processed and displayed as in A. (C) hPASKΔN, cAMP-dependent protein kinase (PKA), and myosin light chain kinase (MLCK) were incubated with their respective substrates and [γ-³²P]ATP in the presence or absence of various amounts of hPASK PAS-A. Reactions were stopped after 5 min, and incorporation of ³²P into substrate was determined by liquid scintillation counting. Incorporation of ³²P into substrate was normalized to hPASKΔN with no added PAS-A and plotted (error bars indicate SD).

hPASK is activated by phosphorylation. (A) Purified recombinant hPASK was incubated at 30°C in the presence of 2 mM [γ-³²P]ATP for the indicated time in the presence or absence of 10 mM EDTA. Samples were resolved by SDS/PAGE followed by silver staining (Upper). Incorporation of ³²P into the hPASK protein was visualized by autoradiography (Lower). (B) Untreated hPASK enzyme and the 10- and 60-min prephosphorylated samples from A were incubated with 0.2 mM AMARA peptide (AMARAASAAALARRR) and 2 mM [γ-³²P]ATP. Reactions were quenched at the times indicated, and incorporation of ³²P into peptide substrate was determined by liquid scintillation counting of purified peptide.