Susan Jaken

Coordination of Three Signaling Enzymes by AKAP79, a Mammalian Scaffold Protein

Multivalent binding proteins, such as the yeast scaffold protein Sterile-5, coordinate the location of kinases by serving as platforms for the assembly of signaling units. Similarly, in mammalian cells the cyclic adenosine 3′,5′-monophosphate-dependent protein kinase (PKA) and phosphatase 2B [calcineurin (CaN)] are complexed by an A kinase anchoring protein, AKAP79. Deletion analysis and binding studies demonstrate that a third enzyme, protein kinase C (PKC), binds AKAP79 at a site distinct from those bound by PKA or CaN. The subcellular distributions of PKC and AKAP79 were similar in neurons. Thus, AKAP79 appears to function as a scaffold protein for three multifunctional enzymes.

Protein kinase C isozymes and substrates

As most cells express more than one type of protein kinase C (PKC), it has been difficult to establish the role of individual PKCs in cellular functions. Isozyme differences in cofactor requirements and subcellular location, in addition to variability in expression of PKCs and substrates among various cell types, are all involved in determining the effects of PKC activation. Recent identification of cellular PKC-targeting proteins and of isozyme-selective functions has provided new insight into the roles of individual PKCs in cellular processes.

Mechanism of action of the phorbol ester tumor promoters: Specific receptors for lipophilic ligands

Cells and tissue preparations specifically bind the phorbol ester tumor promoters. The agreement in structure-activity relationships between binding and biological response strongly argues that these binding sites function as phorbol ester receptors. Upon subcellular fractionation, the phorbol ester binding activity is particulate. In addition, a phorbol ester apo-receptor can be detected in cytosol which requires phospholipids for reconstitution. This apo-receptor appears to correspond to protein kinase C. Diacylglycerols, the probable natural activators of protein kinase C, competitively inhibit phorbol ester binding, consistent with their being the postulated endogenous phorbol ester analogs. In certain systems, heterogeneity of phorbol ester binding is found. An outstanding issue therefore is whether protein kinase C is the phorbol ester receptor or whether it is only the most abundant class of receptor. Although this question remains unresolved, we can demonstrate heterogeneity of phorbol ester binding by reconstitution of apo-receptor into a heterogeneous lipid environment.

35H, a sequence isolated as a protein kinase C binding protein, is a novel member of the adducin family.

We recently cloned a partial cDNA (35H) for a protein kinase C (PKC) binding protein from a rat kidney cDNA library and demonstrated that it is a PKC substrate in vitro (Chapline, C., Ramsay, K., Klauck, T., and Jaken, S.(1993) J. Biol. Chem. 268, 6858-6861). Additional library screening and 5′ rapid amplification of cDNA ends were used to obtain the complete open reading frame. Amino acid sequence analysis, DNA sequence analysis, and Northern analysis indicate that 35H is a unique cDNA related to α- and β-adducins. Antisera prepared to the 35H bacterial fusion protein recognized two polypeptides of 80 and 90 kDa on immunoblots of kidney homogenates and cultured renal proximal tubule epithelial cell extracts. The 35H-related proteins were similar to α- and β-adducins in that they were preferentially recovered in the Triton X-100-insoluble (cytoskeletal, CSK) fraction of cell extracts and were predominantly localized to cell borders. Phorbol esters stimulated phosphorylation of CSK 35H proteins, thus emphasizing that sequences isolated according to PKC binding activity in vitro are also PKC substrates in vivo. The phosphorylated forms of the 35H proteins were preferentially recovered in the soluble fraction, thus demonstrating that phosphorylation regulates their CSK association and, thereby, their function in regulating cytoskeletal assemblies. We have isolated another PKC binding protein partial cDNA (clone 45) from a rat fibroblast library with substantial homology to α-adducin. Antisera raised against this expressed sequence recognized a protein of 120 kDa, the reported size of α-adducin, on immunoblots of renal proximal tubule epithelial cell extracts. A 120-kDa protein that cross-reacts with the clone 45 (α-adducin) antisera coprecipitated with 35H immunecomplexes, indicating that α-adducin associates with 35H proteins in vivo. Taken together, these results indicate that 35H is a new, widely expressed form of adducin capable of forming heterodimers with α-adducin. We propose naming this adducin homologue -adducin.

Signal transduction for proliferation and differentiation in keratinocytes.

In mouse and human epidermis, the Ca2+ environment of the basal cell layer is substantially below serum Ca2+, while that of the granular cell layer is unusually high. Reduction of extracellular Ca2+ concentration (Cao) in the medium of keratinocyte cultures maintains a basal cell phenotype while serum Ca2+ concentrations induce terminal differentiation. Measurements of intracellular Ca2+ (Cai) by the use of Fura 2 and digital imaging technology reveal that Cai increases 10-20-fold in response to an increase in Cao and remains elevated. Concomitant with the rise in Cai is an increase in the metabolism of phosphatidylinositol (PI) to yield inositol phosphates and diacylglycerol. PI metabolism is also stimulated by calcium ionophores suggesting that a rise in Cai is directly responsible. The consequent increase in diacylglycerol and Cai would activate protein kinase C, an event known to trigger epidermal differentiation. Specific Cao and Cai determine the expression of individual markers of keratinocyte differentiation in vitro. These findings may account for the importance of the Ca2+ gradient for maintaining regulated growth and differentiation of the epidermis in vivo.

Increased protein kinase Cδ in mammary tumor cells: relationship to transformation and metastatic progression

Relatively little is known about the molecular mechanisms of tumor promotion/progression in mammary carcinogenesis. Increased protein kinase C (PKC) activity is known to promote tumor formation in several tissues; however, its role in mammary carcinogenesis is not yet known. To determine if individual PKCs may selectively regulate properties of mammary tumor cells, we compared PKC isozyme levels in mammary tumor cell lines with low, moderate and high metastatic potential. All three cell lines expressed α, δ, ε and ζ PKCs; however, PKCδ levels were relatively increased in the highly metastatic cells. To determine if increased PKCδ could contribute to promotion/progression, we overexpressed PKCδ in the low and moderately metastatic cell lines. PKCδ overexpression had no significant effect on growth of adherent cells, but significantly increased anchorage-independent growth. Conversely, expressing the regulatory domain of PKCδ (RDδ), a putative PKCδ inhibitory fragment, inhibited anchorage-independent growth. The efficacy of RDδ as a PKCδ inhibitor was demonstrated by showing that RDδ selectively interfered with PKCδ subcellular location and significantly interfered with phosphorylation of the PKC cytoskeletal substrate, adducin. PKC-dependent phosphorylation of cytoskeletal substrate proteins, such as adducin, provides a mechanistic link between increased PKCδ activity and phenotypic changes in cytoskeletal-dependent processes such as migration and attachment, two processes that are relevant to metastatic potential. The reciprocal growth effects of expressing PKCδ and RDδ as gain and loss of function constructs, respectively, provide strong evidence that PKCδ regulates processes important for anchorage-independent growth in these mammary tumor cells.

Cloning and Characterization of a Glucocorticoid-induced Diacylglycerol Kinase

Diacylglycerol kinase (DGK) plays a key role in cellular processes by regulating the intracellular concentration of the second messenger diacylglycerol. We screened a hamster DDT1 smooth muscle cell library and isolated a unique, glucocorticoid-inducible cDNA with substantial homology to known DGKs. DGK activity was increased in lysates of insect cells infected with recombinant baculovirus containing this cDNA. Antibodies raised against expressed sequences recognized a glucocorticoid-inducible 130-140-kDa protein on immunoblots of DDT1 cell lysates. Thus, this sequence appears to be a new member of the DGK family that we refer to as DGKη. Homology to other DGKs was apparent in domains that are thought to be important for DGK function including the cysteine-rich motifs and potential catalytic domains. DGKη shares substantial homology with DGKδ including the N-terminal pleckstrin homology domain. The tissue distribution of DGKη message (determined by ribonuclease protection assays) and protein (determined by immunoblots) was broader than reported for other DGKs, indicating that DGKη may play a more general role in regulating cellular DG levels than other DGKs. Heterogeneity among DGK family members indicates that individual DGKs may have unique functions.

Protein kinase C and tumor promoters.

The complexity of PKC has made it difficult to define the precise biochemical processes associated with the various PKC-related cellular responses observed. In the past year, we have seen progress in complementary approaches that are helping to solve the puzzles. These include purification and characterization of individual isozymes, expression of normal and mutant PKCs, immunolocalization, and identification of specific activators and inhibitors. All of these will be useful in identifying the primary targets of PKC phosphorylation and their function.

Scatter factor stimulates migration of vascular endothelium and capillary-like tube formation.

Scatter factors (SFs) are heat- and trypsin-sensitive cytokines secreted by fibroblastic and vascular smooth muscle cell lines which stimulate motility of normal epithelium, carcinoma cells, and vascular endothelium. Human and mouse SFs have been purified and identified as 90 kD heterodimeric proteins consisting of heavy (58 kD) and light (31 kD) disulfide-bonded subunits. Partial amino acid sequence data from SF-derived tryptic peptides indicate marked sequence homology with hepatocyte growth factors, suggesting a common multigene family. In this chapter we describe the regulation by SF of vascular endothelial cell chemotaxis and chemokinesis; migration from microcarrier beads to flat surfaces; invasion through porous filters coated with reconstituted basement membrane; secretion of plasminogen activator; and in vitro capillary-like tube formation on a basement membrane surface.

Protein kinase C isozymes and substrates in mammary carcinogenesis.

Protein kinase C (PKC) comprises a family of ubiquitously expressed phospholipid-dependent enzymes that regulate cell growth and differentiation. Several effectors that modify mammary cell biology work at least partially through PKC-dependent pathways. Studies with mammary epithelial cells and tissues have demonstrated probable roles for the PKCs in processes associated with carcinogenesis including proliferation, estrogen sensitivity, and apoptosis. The involvement of PKCs in this wide variety of responses may in part be explained by the expression of multiple PKCs in breast tissue and the possibility that individual PKCs selectively phosphorylate different proteins and preferentially mediate different biological responses. Further understanding of the role of individual PKCs in mammary cell growth and tumor promotion/progression is likely to lead to new insights for breast cancer diagnosis and treatment.