Disruption of AKAP Function by Gene Targeting

Although knockout and RNA strategies have revealed crucial functions of AKAPs in elementary processes whose dysregulation causes disease, it is not clear to which degree the loss of the AKAP function of a protein, i.e. its ability to interact with R subunits of PKA, contributes to the phenotypes because gene knockout deletes all functions of a protein.

Knockout of AKAP149 (also termed AKAP1) decreases fertility in female null mutant mice, but not in heterozygotes (Newhall et al. 2006). Oocytes from null mutant females are either degenerated or arrested in the germinal vesicle state. RIIa does not associate with mitochondria as in wild-type oocytes, suggesting that AKAP149-dependent localization of PKA at mitochondria is essential to drive oocyte maturation.

AKAP4, also termed fibrous sheath component 1, is sperm-specifically expressed and the major fibrous sheath protein of the principal piece of the sperm flagellum. AKAP4 knockout mice are infertile due to inhibition of sperm motility. Immotility is associated with the disruption of the fibrous sheath in spermatozoa and the redistribution of RIIa subunits of PKA from the particulate to the soluble fraction (Huang et al. 2005).

Disruption of D-AKAP2 increases the cardiac response to cholinergic stimuli. The mutant mice suffer from cardiac arrhythmias, and approximately 50% of homozygous and 25% of heterozygous mice die in the first year of life, pointing to a key role of D-AKAP2 in the control of heart rhythm (Tingley et al. 2007).

AKAP150 directly binds L-type Ca2+ channels in neurons (Hall et al. 2007; Oliveria et al. 2007). AKAP150 facilitates PKA phosphorylation of serine 1928 of the aj subunit of the channel upon P-adrenergic stimulation. This is abolished in the knockout mice, but does not cause detectable phenotypic changes of the animal. Consistently, knockin mice with a substitution of serine 1928 for alanine fail to upregulate channel activity in response to adrenergic stimuli (Gao et al. 1997).

WAVE-1, a member of the Wiskott-Aldrich syndrome protein (WASP) family, binds PKA and thus functions as an AKAP. Knockout of WAVE-1 reduces size of the mice and causes behavioural abnormalities, including deficits in sensorimotor function and cognition (Soderling et al. 2003). Approximately one third of the homozygous null progeny die within 24-48 h of birth. WAVE-1 knockout mice are characterised by a phenotype similar to the one observed in patients with a mental disorder, 3p syndrome. Analysis of WAVE-1 knockin mice lacking the interaction domain of WAVE-1 for mental disorder-associated GAP protein (MEGAP/WRP) display a similar phenotype as the knockout animals and the patients suffering from 3p syndrome. In the knockin mice the WAVE-1-dependent interaction of WRP with the small GTPase Rac1 does not occur, and thus WRP does not inactivate Rac1 (Soderling et al. 2002, 2007). The loss of the AKAP function in the knockout model may at least partially contribute to the phenotypic differences between the knockout and knockin models.

Microtubule-associated protein (MAP) 2 is a neuron-specific AKAP. Knockout mice lack gross abnormalities, which is most likely due to the compensation of the loss by MAP1B (Teng et al. 2001). However, several subtle changes in hippocampal neurons are detectable. They include reduction in microtubule density in dendrites and of dendritic length, reduced amounts of catalytic, RIIa and RIIß subunits of PKA in dendrites and reduced CREB phosphorylation in response to forskolin (Harada et al. 2002). A disease with a similar phenotype has not been identified.

Ezrin belongs to the ezrin-radixin-moesin (ERM) protein family. The ERM proteins connect the cytoskeleton with plasma membrane proteins (Bretscher et al. 2002). ERM proteins function as AKAPs binding RI and RII subunits of PKA with low affinity (e.g. KD for binding of ezrin to RII ~30 ||M) compared with other AKAPs (e.g. Kd for binding of RIIß to AKAP185 =20 nM) (Dransfield et al. 1997; Henn et al. 2004). Ezrin knockout causes growth retardation and high mortality. Only ~7% of the animals survive to adulthood. The cause of this early death is unclear. Postnatal day-1 mice show, for example, substantial retardation in the development of photoreceptors, reductions in the apical microvilli of retinal pigment epithelium (RPE) and of Müller cells, and reductions of basal infoldings in RPE cells (Bonilha et al. 2006). In addition, surviving mice suffer severe achlorhy-dria, caused by defects in the formation of canalicular apical membranes in gastric parietal cells (Tamura et al. 2005).

The protein encoded by myeloid translocation gene 8, MTG8, is an AKAP (Fukuyama et al. 2001b). The reciprocal translocation, t(8;21)(q22;q22), occurs almost always in leukaemic cells of patients with acute myeloid leukaemia (AML). The translocation disrupts the AML1 gene on chromosome 21 and the ETO (MTG8) gene on chromosome 8 (Miyoshi et al. 1991; Erickson et al. 1992; Nisson et al. 1992; Fukuyama et al. 2001a). MTG8 knockin mice heterozygous for an allele mimicking the human t(8;21) translocation die in mid-gestation from haemorrhage in the central nervous system and exhibit a severe block in foetal liver haematopoiesis (Yergeau et al. 1997).

Taken together, additional genetically modified animal models, where the PKA-binding domain is deleted in selected AKAPs, are required to understand the role of AKAP-PKA interactions in cellular function. Deletion of other interaction domains within AKAPs will elucidate the function of further direct AKAP-mediated protein-protein interactions.

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