Deficient gene expression in protein kinase inhibitor alpha Null mutant mice

Abstract

Protein kinase inhibitor (PKI) is a potent endogenous inhibitor of the cyclic AMP (cAMP)-dependent protein kinase (PKA). It functions by binding the free catalytic (C) subunit with a high affinity and is also known to export nuclear C subunit to the cytoplasm. The significance of these actions with respect to PKI's physiological role is not well understood. To address this, we have generated by homologous recombination mutant mice that are deficient in PKIalpha, one of the three isoforms of PKI. The mice completely lack PKI activity in skeletal muscle and, surprisingly, show decreased basal and isoproterenol-induced gene expression in muscle. Further examination revealed reduced levels of the phosphorylated (active) form of the transcription factor CREB (cAMP response element binding protein) in the knockouts. This phenomenon stems, at least in part, from lower basal PKA activity levels in the mutants, arising from a compensatory increase in the level of the RIalpha subunit of PKA. The deficit in gene induction, however, is not easily explained by current models of PKI function and suggests that PKI may play an as yet undescribed role in PKA signaling.

FIG. 1

Generation of PKIα knockout mice. (a) Targeting strategy at the PKIα locus. Exon 1 is replaced by the neomycin resistance cassette (neo^r) in a recombinant allele generated by homologous recombination. Probe b was used to identify homologous recombinant ES cells on genomic Southern blots. (b) Southern blot of tail genomic DNA from a litter derived from a heterozygote cross. A restriction digest with BamHI and StuI when hybridized with probe a shows two definitive bands; 5.7 kb indicates the recombinant allele present both in the heterozygote (+/−) and in the knockout (−/−); 4 kb represents the wild-type allele present in the wild-type mice (+/+) and the heterozygotes. (c) Northern blot of total RNA from brain and skeletal muscle from wild-type (WT) and knockout (KO) animals. Probe b was used. Note the absence of the 4.3-kb PKIα transcript in the knockout brain and skeletal muscle (Sk. Mus.).

FIG. 2

Absence of PKI activity in PKIα knockout skeletal muscle. Heat-inactivated tissue extracts from wild-type (WT) and PKIα knockout (KO) skeletal muscle were added to an assay measuring phosphorylation of the PKA substrate, kemptide, by exogenous PKA (C subunit). Kinase activity is expressed as a percentage of the control, where 100% represents kinase activity in the absence of any added tissue extract. The PKI present in wild-type muscle extract almost completely inactivates PKA. Extracts from PKIα knockouts have no significant effect even at the highest concentration of protein, demonstrating a complete absence of PKI activity in PKIα-null skeletal muscle. The error bars represent standard errors of the mean.

FIG. 3

Diminished expression of a PKA-responsive gene in skeletal muscle. Northern blot analysis of PEPCK mRNA levels. A riboprobe made with a PEPCK cDNA fragment as a template was used to probe the Northern blots. Each lane represents RNA from an individual wild-type (WT) or knockout (KO) mouse. The blots were reprobed for GAPDH as a control for RNA loading. (a) PEPCK levels induced by fasting are lower in the knockouts than the wild types. (b) PEPCK expression induced by treating the mice with isoproterenol is decreased in the knockouts relative to the wild-type mice. Note that the GAPDH exposure was longer than for panels a and c. (c) Basal levels of PEPCK examined after refeeding are also lower in the knockouts.

FIG. 4

Phosphorylation of CREB is diminished in PKIα knockout skeletal muscle. Western blot analysis of phospho-CREB levels. Vehicle-treated (C) and forskolin-treated (F) skeletal muscle organ cultures were analyzed at the specified time points for both wild-type (WT) and PKIα knockout (KO) mice. (a) Eight minutes after treatment. Basal levels of phospho-CREB shown in control (C) lanes are lower in knockout than in wild-type muscles. In addition, levels of phospho-CREB in the induced (F) muscles are also lower in the knockout than in the wild type. Below is a Western blot for total CREB with the same samples. Note that total CREB content is constant in all lanes. (b) Eighteen minutes after treatment. The levels of phospho-CREB induced by forskolin are almost back to basal levels, and the difference in phospho-CREB levels between wild-type and knockout muscle is magnified.

FIG. 5

Basal PKA activity is decreased in PKIα knockout skeletal muscle. (a) PKA activity was assayed in skeletal muscle extracts in the absence (− cAMP) and presence (+ cAMP) of 5 μM cAMP to determine basal and total PKA activity, respectively. There is no significant difference in the total kinase activity between wild-type (WT) and knockout (KO) mice. (b) Basal activity, in the absence of exogenous cAMP, is shown with an expanded axis. The knockout extract has significantly lower basal activity than the wild type (∗∗, P = 0.003; t test). The error bars represent standard errors of the mean.

FIG. 6

Compensatory increase in RIα regulatory subunit. Muscle extracts from wild-type (WT) and knockout (KO) mice were subjected to Western blotting and probed for the RIα, RIIα, and Cα subunits of PKA. Each lane corresponds to a tissue homogenate from a separate animal. Specific up-regulation of the RIα regulatory subunit (1.6-fold) is seen in the knockouts, with no change in the level of either the RIIα subunit or the C subunit.