Targeting of PKA in mammary epithelial cells. Mechanisms and functional consequences.

Targeting of protein kinases, promoting association with specific partner-molecules and localisation to particular sites within the cell, has come to be recognised as a key mechanism for attributing specificity to these enzymes. In mammary epithelial cells, the repertoire of acute regulatory roles played by cyclic AMP-dependent protein kinase (PKA) differs from that in other lipogenic cell-types. Furthermore, PKA is implicated in the regulation of mammary-specific function, mediating a tonic stimulation of the flux of newly-synthesised casein through its basal secretory pathway. Both these observations imply mammary-specific properties of either PKA targeting systems or of PKA itself. Evidence for the latter is currently lacking. Pulse-chase labelling experiments in the presence and absence of selective effectors of PKA have enabled the site(s) of action of this protein kinase on casein secretion to be localised to the early stages of the secretory pathway. Possible mechanisms are considered for the physical targeting of PKA to the membrane-enclosed components of the secretory pathway and evidence for their occurrence in mammary epithelial cells is presented.

Expression of enzymes of covalent protein modification during regulated and dysregulated proliferation of mammary epithelial cells: PKA, PKC and NMT.

Three proteins are functionally interlinked in the targeting of protein phosphorylation catalyzed by the C-subunit of PKA: PKA itself, AKAPs and NMT. Furthermore, in a variety of biological contexts, mechanisms exist whereby PKA and PKC are able to modulate the activity of one another. We have investigated the expression and subcellular distribution of these proteins in two models of mammary cell proliferation and differentiation--the normal rat mammary gland during pregnancy and lactation and human breast tissue before and after malignant transformation. Modulation of PKA does not acutely affect activity or sub-cellular distribution of PKC in mammary acini, nor does modulation of PKC acutely affect PKA activity or subcellular distribution. Therefore, the co-ordinate expression of these two protein kinases in normal and cancerous mammary epithelial cells and the greater basal activation level of them both accompanying increased mitogenic activity, which we have reported, does not result from short-term cross-talk between them. Although basal and total levels of PKA diminish in rodent mammary epithelial cells during the transition from proliferative to secretory functional mode, the level of expression of AKAPs increases. The expression of two apparently mammary-specific and mostly membrane-associated AKAPs is tightly linked to the onset and maintenance of differentiated function in rat mammary tissue. Paradoxically, the probable analogues of these two AKAPs in human mammary tissue are hyperexpressed when normal epithelial cells transform to a cancer phenotype--conventionally regarded as a process involving a degree of dedifferentiation. Mammary AKAP hyperexpression in breast cancers is accompanied by increases in the levels of total and basal PKA. One mechanism whereby NMT is targeted to membranes, via interaction with ribosomal proteins, has recently been elucidated. Our data support the contention that the localization of NMT is an important variable in the regulation of cellular proliferation, but they do not characterize the mechanisms whereby the differential targeting of NMT is achieved. As yet we lack a full tool-kit with which to examine NMT either to draw firm conclusions regarding the identity of particular isoforms found in particular sub-cellular locations or to define the relationships between these different molecular variants. However, it is technically possible to transfect cells with inducible NMT expression constructs engineered in such a way that the recombinant, catalytically competent, NMT that they encode is targeted either to membranes or to cytosol: an exploration of the effects of such transfections on cellular proliferation would afford a critical test of the mechanistic involvement of NMT in the control of mitogenesis.

Organization and alternative splicing of the Caenorhabditis elegans cAMP-dependent protein kinase catalytic-subunit gene (kin-1).

The cAMP-dependent protein kinase (protein kinase A, PK-A) is multifunctional in nature, with key roles in the control of diverse aspects of eukaryotic cellular activity. In the case of the free-living nematode, Caenorhabditis elegans, a gene encoding the PK-A catalytic subunit has been identified and two isoforms of this subunit, arising from a C-terminal alternative-splicing event, have been characterized [Gross, Bagchi, Lu and Rubin (1990) J. Biol. Chem. 265, 6896-6907]. Here we report the occurrence of N-terminal alternative-splicing events that, in addition to generating a multiplicity of non-myristoylatable isoforms, also generate the myristoylated variant(s) of the catalytic subunit that we have recently characterized [Aspbury, Fisher, Rees and Clegg (1997) Biochem. Biophys. Res. Commun. 238, 523-527]. The gene spans more than 36 kb and is divided into a total of 13 exons. Each of the mature transcripts contains only 7 exons. In addition to the already characterized exon 1, the 5'-untranslated region and first intron actually contain 5 other exons, any one of which may be alternatively spliced on to exon 2 at the 5' end of the pre-mRNA. This N-terminal alternative splicing occurs in combination with either of the already characterized C-terminal alternative exons. Thus, C. elegans expresses at least 12 different isoforms of the catalytic subunit of PK-A. The significance of this unprecedented structural diversity in the family of PK-A catalytic subunits is discussed.

Casein secretion in mammary tissue: tonic regulation of basal secretion by protein kinase A.

Despite its quantitative importance in the secretion of lactoproteins, little is known about the triggering and control mechanisms that initiate, regulate and terminate the operation of the basal pathway of lactoprotein secretion throughout the lactation cycle. This study investigated the possible modulation by cAMP-mediated mechanisms, of cellular transit of newly-synthesised caseins and their basal secretion in explants of mammary tissue from lactating rats and rabbits. Enhancement of the rate of secretion of newly-synthesised caseins occurs when mammary explants are challenged in vitro with agents that activate protein kinase A (PKA). Inhibition of PKA slows casein secretion. The PKA-sensitive step(s) in casein secretion is early in the exocytosis pathway but inhibition of PKA does not impair casein maturation. Ultrastructural, immunochemical and biochemical methods locate PKA on membranes of vesicles situated in the Golgi region. Exposure of tissue to a cell-permeant PKA inhibitor results in morphological modification of these vesicular structures. We conclude that PKA mediates tonic positive regulation of the basal secretory pathway for lactoproteins in the mammary epithelial cell.

N-Myristoylation of the catalytic subunit of cAMP-dependent protein kinase in the free-living nematode Caenorhabditis elegans.

N-Myristoylation of the catalytic subunit (C-subunit) of cAMP-dependent protein kinase is widespread in animal cells. Some invertebrates express non-myristoylated isoforms of C-subunit but these co-exist with at least one myristoylated isoform. The generality of this observation implies an indispensable function for myristoylated C-subunit, but notwithstanding this, neither of the C-subunit isoforms hitherto described in C. elegans is apparently N-myristoylated. In light of this anomaly, the myristoylation status of the C-subunit has been examined in adult C. elegans. Evidence is presented for the presence of an N-myristoylated isoform.

Elevation of protein kinase A and protein kinase C activities in malignant as compared with normal human breast tissue.

Because of their central role in the transduction of extracellular signals, protein kinases A (PKA) and C (PKC) are critical enzymes in the control of cellular proliferation and differentiation. We have measured the catalytic activity of PKA and PKC, as well as the regulatory subunit expression for PKA, in paired samples of normal and malignant breast tissue from 13 patients with breast cancer. Paired non-parametric (Wilcoxon) analysis revealed significantly higher values for both basal (P = 0.0002) and total (P = 0.0002) PKA catalytic activity in malignant compared with normal breast in all 13 paired tissue samples. Expression of both R1- and RII-PKA regulatory subunits were also higher in malignant tissue from 12 (P = 0.0005) and 9 (P = 0.01) of the 13 pairs, respectively. However, the degree of RI-subunit overexpression in malignant tissue was greater than that of the RII-subunit, as demonstrated by an increase in the RI/RII subunit ratio in 10 of the 13 paired samples (P = 0.017). Total PKC catalytic activity was elevated in 11 of the 13 malignant tissue specimens when compared with corresponding normal breast tissue (P = 0.01). This was accounted for by an increase in Ca(2+)-dependent PKC activity (P = 0.01), there being no significant increase in Ca(2+)-independent PKC activity. These data suggest that the activities of both PKA and PKC signalling pathways are intrinsically higher in malignant compared with normal breast tissue and these may therefore represent targets for interventive treatment of breast cancer.

Responses of glucose metabolism to insulin in perfused mammary tissue of lactating rats: influence of dietary history and recent insulin experience.

Mammary tissue of lactating rats has been perfused in situ to measure the effects of insulin and of previous dietary history on utilization of glucose and its incorporation into lipid. This experimental model allows the direct effects of insulin on mammary tissue to be characterized without influence from the secondary consequences of insulin administration that may accompany treatment of the intact animal with this hormone. In mammary tissue from rats starved for 24 h, glucose utilization was stimulated by insulin treatment. The threshold for insulin response was between 0.01 and 0.02 mU ml-1, and no further stimulation took place between 0.02 and 0.04 mU ml-1 insulin. The maximum response was reached within 50 min of the onset of insulin challenge and remained at a plateau value regardless of whether insulin was continuously present or was withdrawn after 15 min. Incorporation of glucose into lipid contributed to this glucose uptake. When no insulin was present in the perfusate, the rate of this process was around 3-fold greater in perfused mammary tissue from fed lactating rats than from those starved for 24 h. Insulin accelerated lipogenesis from glucose approximately 3- and 2-fold, respectively, in these two experimental groups. As for glucose uptake, the role of insulin in the regulation of mammary lipogenesis appeared to be to 'prime' the tissue and commit it to a response that was subsequently insulin independent.

Separation of H7-resistant protein kinase C isoforms from gonadotroph-derived alpha T3-1 cells.

Certain physiological events in anterior pituitary tissue are regulated by protein kinase C that is relatively resistant to the inhibitor, H7. In the present studies we have shown that two H7-resistant protein kinase C activities may be isolated from (pituitary) gonadotroph-derived alpha T3-1 cells using hydroxyapatite chromatography. These activities have the characteristics of PKC zeta and an apparently novel PKC isoform. The availability of clonal alpha T3-1 cells will facilitate investigations of the pituitary events.

Phorbol ester and diacylglycerol activation of native protein kinase C species from various tissues.

The characteristics of PKC activation induced by a number of compounds were investigated using PKCs, partially-purified from sources with a naturally high abundance of certain Ca2+ dependent PKC isoforms. Native isoforms were used rather than PKC isoforms expressed from a baculovirus system to assess the effect of tissue specific factors on activity. However, some data using recombinant PKC alpha were included for comparison. The presence of specific PKC isoforms in different tissues was determined using Western blot analysis. Protein kinase C alpha, beta 1, delta, epsilon, and zeta/iota were all present in rat midbrain cytosolic extract, PKC alpha, beta 1, delta, and zeta/iota were present in spleen cytosol, and PKC alpha and zeta/iota were present in COS 7 cell cytosol. The predominance of alpha and beta activities in COS 7 and spleen extracts respectively was confirmed by enzymic assay. The PKC activity assay was configured such that the Ca2+ dependence of the PKC activity induced by different PKC activators could be determined. Phorbol 12,13-dibutyrate (PDBu) was virtually equipotent on the Ca(2+)-dependent PKC activity from midbrain and spleen and slightly less potent on that from COS 7 cells. In the absence of Ca2+, PDBu was considerably less potent overall (as, indeed, were the other PKC activators) and was less potent on COS 7 cell PKC than on those from midbrain or spleen. Mezerein was more potent than PDBu at inducing PKC activity in COS 7 cell extracts in either the absence or presence of Ca2+ whereas in the presence of Ca2+, mezerein was slightly less potent on midbrain and spleen than PDBu and equipotent in the absence of Ca2+. Maximum values for Ca(2+)-independent activation by mezerein indicated that this activator was particularly effective in recruiting Ca(2+)-dependent PKC isoform activity in a Ca2+ free environment. The greater potency of mezerein on PKC alpha was confirmed using PKC alpha and beta further purified from rat spleen by hydroxylapatite (HAP) chromatography. The effects of both PDBu and mezerein were investigated using anterior pituitary tissue where a particularly high potency of mezerein in the absence of Ca2+ was noted. The diacylglycerol, 1,2-dioctanoyl-sn-glycerol (DOG), appeared to cause little or no activation of native Ca(2+)-dependent isoforms in Ca2+ free conditions unlike another longer chain diacylglycerol, 1,2-dioleoyl-sn-glycerol. Also DOG activated midbrain PKCs more potently than PKCs from spleen or COS 7 cells (or lung and pituitary tissue) in the presence of Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)