Amino Acids

CD C

TD C

Fig. 1 Peptide substitution array of the RII-binding domain of AKAP18S. Shown is a peptide array of spot-synthesized 25-amino-acid-long peptides, comprising the RII-binding domain of AKAP18S (positions 296-320; vertical) in which every amino acid is replaced by the amino acids indicated at the top of the membrane. Peptides were spot-synthesised on cellulose membranes and probed for binding to 32P-labelled RII subunits of PKA (RII-overlay assay). Binding of RII-subunits was detected by autoradiography. Amino acids indicated by solid boxes are conserved in RII-binding domains. Introduction of proline into the core RII-binding domain leads to disruption of the a-helical structure of the domain, which reduces or abolishes RII-binding (highlighted by the dotted box). Amino acids are indicated by single letter code structure of RII-binding domains was initially predicted from a fragment of AKAP-Lbc, Ht31 (Carr et al. 1991) and confirmed by NMR studies for a peptide (Ht31) derived from the RII-binding domain of AKAP-Lbc (Table 1) and for a peptide derived from the RII-binding domain of AKAP79 (Newlon et al. 1999). Recent X-ray crystallography studies confirmed the amphipathic helix structures for AKAPIS (see below) and a peptide derived from the RII-binding domain of D-AKAP1 (Gold et al. 2006; Kinderman et al. 2006).

The dissociation constants for the interactions of the peptide Ht31 with RIIa and RI subunits are KD = 1.3 ± 0.06 |M and 2.2 ± 0.03 nM, respectively (Table 1) (Carr et al. 1992; Alto et al. 2003). Consistently, in vitro and cellular assays revealed that the peptide Ht31 functions as an effective disruptor of both AKAP-RI and AKAP-RII

ARNDQEGHI LKMFPSTWYV

ARNDQEGHI LKMFPSTWYV

Fig. 1 Peptide substitution array of the RII-binding domain of AKAP18S. Shown is a peptide array of spot-synthesized 25-amino-acid-long peptides, comprising the RII-binding domain of AKAP18S (positions 296-320; vertical) in which every amino acid is replaced by the amino acids indicated at the top of the membrane. Peptides were spot-synthesised on cellulose membranes and probed for binding to 32P-labelled RII subunits of PKA (RII-overlay assay). Binding of RII-subunits was detected by autoradiography. Amino acids indicated by solid boxes are conserved in RII-binding domains. Introduction of proline into the core RII-binding domain leads to disruption of the a-helical structure of the domain, which reduces or abolishes RII-binding (highlighted by the dotted box). Amino acids are indicated by single letter code

Table 1 Peptides disrupting AKAP-dependent protein-protein interactions

Peptide

RI

mean ± SEM) RII

Properties

Ht31

DLIEEAASRIVDAVIE-QVKAAGAY

1.300 ±6.0

4.0 ± 1.2a

Global disruptor

2.2 ± 0.03"

Ht31-P

DLIEEAASRPVDAVPE-QVKAAGAY

n.b.

n.b.

Neg. control

A KB (dual)

VQGNTDEAQEELAWKIAKMIVSDVMQQ

48 ±4

2.2 ± 0.2

Global disruptor"

A KB (RII)

VQGNTDEAQEELLWKIAKMIVSDVMQQ

2.493 ± 409

2.7 ±0.1

RII-prefeningc

A KB (RI)

FEELAWKIAKMIWSDVFQQ

5.2 ± 0.5

456 ± 33

RI-preferringc

A KB (null)

VQGNTDEAQEELAWKIEKMIWSDVMQQ

998 ± 66

>10.000

Neg. control0

AKAPIS

QIEYLAKQIVDNAIQQA

0.23 ± 0.05

0.45 ± 0.07

Global disruptor''

AKAP188-wt-pep.

PEDAELVRLSKRLVENAVLKAVQQY

n.d.

0.4 ± 0.3

Global disrupto^

AKAP185-L314E

PEDAELVRLS KRLVENAVE KAVQQ Y

n.d.

0.7 ± 0.5

Global disrupto^

AKAP185-PP

PEDAELVRLS KRLPENAPLKAVQQY

n.d.

n.b.

Neg. controld

RIAD

LEQYANQLADQIIKEATE

1.0 ±0.2

1.760 ±290

RI-preferringe

Arg9-ll-PLN

RRRRRRRRRRRASTIEMPQQ

n.a.

n.a.

AKAP18S-PLN disruptor1

AKAP15-LZ

ENAVLKAVQQYLEETQN

n.a.

n.a.

AKAP18a-L type Ca2+ channel disruptor8

AKAP-derived PKA-anchoring disruptor peptides binding both regulatory RI and RII subunits of PKA globally inhibit PKA anchoring to cellular compartments. As indicated, a few peptides preferentially uncouple PKA RI subunits from AKAPs since their binding affinities for RI subunits are higher than for RII subunits. Examples of peptides disrupting other AKAP-dependent protein-protein interactions are also listed. In addition, several proline (P)-containing inactive control peptides are shown (e.g. Ht31-P for experiments with Ht31). KD, equilibrium dissociation constants; n.b., no binding; n.d., not detected; n.a., not available a Carr et al. (1991) "Alto et al. (2003) cBurns-Hamuro et al. (2003) dHundsrucker et al. (2006) e Carlson et al. (2006) 'Lygren et al. (2007) 8 Hulme et al. (2002)

interactions. Alto et al. (2003) utilized peptides derived from RII-binding domains of AKAP-KL, AKAP79, mAKAP, AKAP18a and AKAP-Lbc for amino acid substitution analysis at all positions within the peptides and a bioinformatics approach to develop a high-affinity peptide termed AKAP in silico (AKAPIS). The dissociation constants for the binding of AKAPIS to regulatory RI and RII subunits of PKA are Kd = 0.23 ± 0.05 nM and 0.45 ± 0.07 nM, respectively (Table 1). Consistent with this, AKAPIS disrupts AKAP-RI and AKAP-RII interactions. In HEK293 cells overexpressing glutamate GluRl receptors, AKAPIS displaces PKA type II from the receptors and thereby evokes a rapid reduction of glutamate GluRl receptor currents. Among the AKAPs binding RII subunits with high affinity is AKAP185 (RIIa: KD = 31 nM; RII|: KD = 20 nM) (Henn et al. 2004). Truncated versions of AKAP185 bind RII subunits with even higher affinity than the full-length protein (e.g. KD of amino acids 124-353 for binding RII| = 4 nM; Henn et al. 2004). This observation led to the development of 25-amino-acid-long PKA-anchoring disruptor peptides derived from the RII-binding domain of AKAP185. The wild-type peptide AKAP185-wt binds RIIa subunits (KD = 0.4 ± 0.3 nM) with higher affinity than peptides derived from the RII-binding domains of other AKAPs and with a similar high affinity as AKAPIS (Table 1). Peptide substitution arrays and Biacore measurements revealed that several AKAP185-derived peptides bind RII subunits with a similar high affinity (Fig. 1). One of these is the peptide AKAP185-L314E, characterised by greater water solubility than AKAP185-wt. Therefore, it is widely applicable for the study of compartmentalized PKA signalling. The ability of AKAP185-derived peptides to displace RI subunits from AKAPs has not been investigated.

The dual specificity AKAPs D-AKAP1 and D-AKAP2 bind both RI and RII subu-nits of PKA. This observation initiated the development of peptides selectively disrupting AKAP-RI and AKAP-RII interactions. On the basis of the PKA-anchoring domain of D-AKAP2, Burns-Hamuro et al. (2003) generated the peptide AKB-RI (A-kinase-binding-RI. It binds RIa subunits with approximately 90-fold higher affinity than RIIa subunits (Kd = 5.2 ± 0.5 nM versus 456 ± 33 nM) and thus preferentially disrupts AKAP-RI interactions. In addition, the peptide AKB-RII with opposite binding characteristics was developed (Burns-Hamuro et al. 2003). It binds RIa subunits with almost 1,000-fold lower affinity than RIIa subunits (KD = 2.4 ± 0.4 |M versus 2.7 ± 0.1 nM). Carlson et al. (2006) developed the peptide RIAD (RI-Anchoring Disruptor). It was optimised for RI binding based on the PKA-anchoring domains of the dual-specificity AKAPs D-AKAP1, AKAP149, AKAP82 and ezrin, which possess higher binding affinity for RIa than for RIIa subunits. The dissociation constants for the binding of RIAD to RIa and RIIa subunits of PKA are KD = 1.0 ± 0.1 nM and 1.76 ± 0.3 |M, respectively (Table 1). Compared with other peptides, RIAD is, therefore, the peptide with both the highest affinity and specificity for RIa subunits. Consequences of disruption of AKAP-RI interactions with RIAD in cultured T-cells are reduced phosphorylation and therefore inactivation of C-terminal Src kinase (Csk) by PKA type I. This in turn leads to a reduced phosphorylation of the lymphocyte-specific protein-thyosine kinase (Lck), which causes up-regulation of T cell receptor signalling. RIAD also reduced progesterone production in adrenocortical cells, which thus depends on the interaction of RI with AKAPs (Carlson et al. 2006).

Several PKA-anchoring disruptor peptides have been rendered cell-permeable (e.g. coupling Ht31 or AKAP185-derived peptides with stearate) and utilised to study functions of AKAP-PKA interactions in cultured cells (Table 2). Moita et al. (2002) carried out experiments with stearate-coupled Ht31 peptide in brains of rats. They infused the peptide into the lateral amygdala in order to examine the role of PKA anchoring in auditory fear memory. Behavioural tests revealed that anchoring of PKA is necessary for the consolidation, but not for the acquisition of conditioned fear.

A few peptides have been used to displace interaction partners other than PKA from AKAPs. For example, disruption of the interaction of AKAP185 with PLN employing a peptide derived from the PLN interaction site for AKAP185 reduces the velocity of Ca2+ reuptake into the SR of cardiac myocytes (Lygren et al. 2007). The effect of the peptides resembles the effect of RNAi directed against AKAP185 (see above). A peptide derived from the leucine zipper motif of AKAP18a that interferes with the interaction of AKAP18a with L-type Ca2+ channels prevents P-adrenoceptor-mediated increases of Ca2+ entry into cardiac myocytes (Hulme et al. 2003).

The properties of the peptides described here are summarized in Table 1. Functions of direct AKAP-mediated protein-protein interactions identified by the use of PKA-anchoring disruptor peptides and peptides disrupting interactions of AKAPs with other partners are listed in Table 2.

0 0

Post a comment