1.  INTRODUCTION

    Targeting of protein kinases is a mechanism of imposing functional selectivity on their action, additional to that which their own sequence-recognition specificity gives them. Furthermore, the characteristics of an individual targeting mechanism may be such as to enable a single enzyme to manifest diverse specificities both between cells and, within a single cell, at different points in time or during a programme of growth and differentiation.

1.1 PKA isozyme diversity

     Cyclic AMP-dependent protein kinase (PKA) is one of a relatively small number   of generic   protein   kinases   acting   pleiotropically    as   primary transducers of regulatory signals within cells. It is catalytically activated in proportion  to  the  prevailing   concentration   of cAMP   in  its   immediate environment. In its inactive state, PKA exists as a heterotetramer consisting of two catalytic (C-) and two regulatory (R-) subunits. Multiple isozymes of both R- and C- subunit are encoded by different genes (RIa,(3; RIIa,(3; Ca,(3. Splice variants of C-subunit also exist, most strikingly in C. elegans but also in mammalian species. The precise function of all this diversity is not known with certainty either in C. elegans or in mammals, but evidence is beginning to emerge to support the suggestions that determinants of targeting   and/or   stability   and   turnover   of the   C-subunit   protein molecule may reside in these alternative terminal sequences.

1.2  Basal activity and activation of PKA     
 
      The presence of R-subunit sequences occluding the substrate cleft of C-subunit causes the latter to remain catalytically inactive in the holoenzyme. The cAMP signal is transduced via its high-affinity binding to two sites on each R-subunit, causing them to dissociate as an R-subunit dimer, concomitantly liberating two catalytically active C-subunits. Phosphorylation of proteins by C-subunit can lead to a variety of biological effects depending on the identity of the phospho-acceptor substrate protein. Examples of these include: enzymes catalysing biosynthesis, degradation and metabolic interconversion; transcriptional regulators; ion channels; transmembrane receptors and other signalling mediator proteins. The PKA activation cycle is reversed by the action of cAMP phosphodiesterases. When making experimental measurements of PKA activity, a “snapshot” of the dissociation status at the time of sampling can be made by assaying in the absence of added cAMP to determine the “basal” or “expressed” activity. The “total” activity (measured in the presence of a saturating excess of cAMP) measures the maximum potential of the tissue for C-subunit catalysed protein kinase activity in the event of complete dissociation of all the cellular holoenzyme. In real situations within living cells, activation of PKA is not an “all-or-none” phenomenon but a proportional response to the prevailing cAMP level which may itself vary from place to place within the cell .   Thus the tonic  or basal level  of PKA activation is  an important determinant of cellular activity (see e.g. Thorens et al.). This function of PKA has attracted much less attention than its complementary role in the mediation of cellular responses of cells to acute modulators of cAMP levels, such as β-adrenergic agents or glucagon.

     1.3 PKA targeting       

          The biological effects of PKA can be modulated by restricting its distribution within the cell. One way in which this can be achieved is by the expression of AKAPs (A-kinase anchoring proteins) which bind to R-subunits and to a secondary binding partner, thereby tethering the holoenzyme to that secondary partner. Cells express a number of AKAPs, and a temporal dimension can be added to the spatial regulation of PKA if various AKAPs (having specificity for different secondary binding partners) are differentially expressed with respect to time, for instance, throughout the cell cycle or during differentiation. In addition to holoenzyme targeting via AKAPs, it is possible that spatial localisation of PKA catalytic activity can be achieved by intrinsic targeting of C-subunit. The myristoylated amino terminus of C-subunit is a candidate motif for involvement in intrinsic targeting. Although the majority of free C-subunit molecules are uniformly distributed throughout the cytosol of most cells, several examples of the specific interaction of C-subunit with partner proteins (in addition to R-subunit and the specific heat-stable inhibitor protein isoforms) have recently been described and atypical sub-cellular distribution of C-subunit has been reported in ovine spermatozoa which express a non-myristoylated splice-variant of the kinase.

2.  RESULTS AND DISCUSSION      

    In mammary epithelial cells, three separate lines of evidence point towards sequestration/targeting of PKA. The first of these concerns the regulation of acetyl-CoA carboxylase (ACC). The phosphorylation cascade controlling its activity is normally initiated by PKA activation; however, this is abrogated in intact mammary epithelial cells although it operates efficiently with intrinsic mammary components in cell-free systems. Secondly, there is evidence for the membrane-localisation of a small fraction of the total mammary cellular PKA. Thirdly, the tonic level of PKA activation in mammary epithelial cells has been shown to modulate the rate of the constitutive pathway of casein secretion and this effect has been localised to early steps (ER-Golgi; Golgi-TGN) in the pathway. The effect of inhibition of basal PKA activity on casein secretion is illustrated in Fig. 1.

                                                       

Fig.1. Suppression of constitutive secretion of newly-synthesised caseins in explants of rat mamma y gland by inhibition of PKA. SDS PAGE analysis of pulse-labelled proteins.

2.1  Association of PKA and AKAPs with membranes of secretory pathway elements
     
     The occurrence of PKA in unfiactionated (microsomal) membranes from unstimulated lactating rat mammary tissue has been confirmed by three independent methods: (1) Western blotting with anti-C-subunit antibodies; (2) photoaffinity-labelling of R-subunits with 8-azido-[³²P]cAMP; (3) enzymic assay of catalytic activity. Following stimulation of adenylate cyclase in intact cells with forskolin or isoprenaline, or direct activation of PKA with cpt cAMP, the following were found to be increased in microsomal membranes: PKA activity (total and expressed components); concentration of total C-subunit; concentration of MI-subunit (Fig. 2). Facultative targeting via conventional AKAPs is implied by this selective sequestration of RII subunit.

       The AKAP content of microsomes from lactating rat mammary tissue was investigated by overlay (ligand blotting) with ³²P-labelled recombinant RII∀ subunit. Major AKAPs with apparent molecular weights of 129000, 106000, 75000 were detected along with endogenous RII subunit at 54000. As previously described, expression of the 129000 and 75000 AKAPs was tightly developmentally regulated: they were expressed during lactation but not during pregnancy nor beyond 24h of involution following induction of milk stasis by litter-removal. AKAP distribution between cytosol and membranes was unaffected by PKA activation in intact cells (Fig. 2).                        

Fig.   2.   Membrane-associated   components   of the  PKA   system   in   mammary  tissue   from lactating rats: effects of PKA activation before tissue fractionation.

       Microsomal membranes were further fractionated on the basis of their buoyant density in sucrose gradients. Scanning down these gradients, AKAPs of M_r 106000, 75000 and intrinsic RII predominated in light membrane fragments enriched in plasma membrane markers. RII was not found in denser membrane fragments, whereas 106000 and 75000 components persisted into fragments of intermediate density, enriched in Golgi membrane markers, but not into denser regions of the gradient. AKAP 129000 began to appear in Golgi/secretory vesicle-enriched fragments and became the predominant AKAP in the denser ER-derived membrane fragments. ER membrane was also, on the basis of protein content, the most abundant component in the gradient. Assay of PKA catalytic activity intrinsic to these membrane fractions, using Kemptide (a synthetic peptide phospho-acceptor substrate) showed firstly that “expressed” activity was virtually absent from all fractions and secondly that “total” activity distributed in a peak coincident with ER membranes. Potential phospho-acceptor substrate proteins of PKA resident in individual membrane fractions were identified by in vitro incubation of membranes (20µg protein) with [(-³²P]ATP in the absence of cAMP. Multiple phospho-proteins were found, and although only their M_r and not their identity was revealed in these experiments, it is noteworthy that all phosphorylations were suppressed by the addition of PKI (a peptide inhibitor selective for PKA) and were only modestly enhanced by the addition to each incubation of 5µg of pure recombinant C-subunit.

3.  CONCLUSIONS AND PERSPECTIVES
      
        These results indicate the presence of two populations of C-subunit molecule  in  mammary  microsomal   membranes.  In   one,  C-subunit  is stoichiometrically associated with R-subunit in the form of heterotetrameric holoenzymes; these are totally cAMP-dependent in their Kemptide kinase activity and are probably anchored predominantly to ER membranes via AKAP 129000. The association of this AKAP with ER membranes, and its apparent molecular size, suggest that it may be the ER-targeted (N1 splice-variant) derivative of the carboxyl-extended D-AKAP 1 splice isoform 1 c (known in rodents as AKAP 121), hitherto thought to be expressed only in live. The other population consists of free C-subunit, occluded from access to Kemptide but able to catalyze the cAMP-independent phosphorylation of membrane-resident proteins that are themselves, at best, poorly accessible to exogenous free C-subunit. These endogenous free C-subunit molecules are tethered to the membrane via an intrinsic targeting mechanism relying only on the ability of C-subunit itself to interact with membrane component(s) – protein or lipid. The hypothesis that terminal domains of C-subunit are involved in its intrinsic targeting has been considered above. Future characterisation of this membrane-associated sub-population will further test this hypothesis and elucidate the mechanism of intrinsic targeting of C-subunit.