Bryostatin 1 Induces Biphasic Activation of Protein Kinase D in Intact Cells

Abstract

Bryostatin 1 and phorbol esters are both potent activators of protein kinase C (PKC), although their specific biological effects can differ in many systems. Here, we report that bryostatin 1 activates protein kinase D (PKD), a novel serine/threonine protein kinase, in intact Swiss 3T3 cells and secondary mouse embryo fibroblasts and in COS-7 cells transiently transfected with a PKD expression construct. The dose response of PKD activation induced by bryostatin 1 follows a striking biphasic pattern with maximal activation achieved at a concentration of 10 nM. Higher concentrations of bryostatin 1 (100 nM) reduced PKD activation induced by phorbol 12,13-dibutyrate to levels stimulated by bryostatin 1 alone. Bryostatin 1-induced PKD activation was markedly attenuated by treatment of cells with the PKC inhibitors bisindolylmaleimide I and Ro 31-8220. However, these agents did not inhibit PKD activity when added directly to in vitro kinase assays, suggesting that bryostatin 1 stimulates PKD activation through a PKC-dependent pathway in intact cells. Our results raise the possibility that activated PKD in intact cells could mediate some of the multiple biphasic biological responses induced by bryostatin 1.Bryostatin 1 is a natural macrocyclic lactone with potent antineoplastic properties in a variety of animal models (1-3) and has entered clinical trials as a potential therapeutic agent (4, 5). Bryostatin family members bind to and activate classic and novel isoforms of protein kinase C (PKC) 1 (6 -9), the major cellular targets of the tumor-promoting phorbol esters (10). Despite appearing to bind to the same cellular targets, the biological responses induced by bryostatin 1 frequently differ from those induced by phorbol esters. For example, many bryostatin 1-mediated effects have unusual characteristics such as biphasic dose-response relationships, delayed kinetics, and the ability to inhibit phorbol ester-induced responses (7,(11)(12)(13)(14)(15)(16)). The precise mechanism(s) by which bryostatin 1 induces these biological effects remain poorly understood.The newly identified protein kinase D (PKD) is a mouse serine/threonine protein kinase with distinct structural features and enzymological properties (17)(18)(19). In particular, the catalytic domain of PKD, which is distantly related to Ca 2ϩ -regulated kinases, shows little homology to the highly conserved regions of the kinase domain of the PKC family. As a consequence of this, PKD does not phosphorylate a variety of known PKC substrates, indicating that PKD has a distinct substrate specificity (18, 19).The amino-terminal region of PKD contains a putative transmembrane domain, two cysteine-rich zinc finger-like motifs, and a pleckstrin homology domain. Unlike all known PKC isoforms, PKD does not contain a pseudosubstrate motif upstream of the cysteine-rich region, and the sequence separating the cysteine-rich repeats of PKD (95 amino acids) is substantially longer than that of classical and novel PKCs (28 and 35 amino acids, respectively). Additionally, residue...

Bryostatin-1: Pharmacology and Therapeutic Potential as a CNS Drug

“…Downregulation is most significant when the PKC isozymes are exposed to high and/or prolonged high concentrations of bryostatin-1. It should be, however, mentioned here that modulation of PKC activity may not be the only biological action of bryostatins (25). For example, in B16/F10 melanoma cells (26) epi-bryostatin-1, a bryostatin-1 stereoisomer, with much lower affinity for PKC, exhibits the same potency in inhibiting cell growth as bryostatin-1 (41), suggesting the involvement of aPKC activity-independent action.…”

Protein Kinase D Potentiates DNA Synthesis and Cell Proliferation Induced by Bombesin, Vasopressin, or Phorbol Esters in Swiss 3T3 Cells

“…Treatment with PKC-selective inhibitors prevented PKD activation by all these factors (22,(31)(32)(33). Furthermore, cotransfection of PKD with constitutively active mutants of PKC ⑀ and dramatically increased the catalytic activity of PKD (24,31) and led to complex formation between PKD and PKC (24).…”

“…PKD can be activated within intact cells by pharmacological agents like biologically active phorbol esters and cell-permeant DAGs as well as by physiological stimuli including GPCR agonists, growth factors, and antigen-receptor engagement (31)(32)(33)(34)(35)(36)(37)(38)(39). Treatment with PKC-selective inhibitors prevented PKD activation by all these factors (22,(31)(32)(33).…”

Identification of in Vivo Phosphorylation Sites Required for Protein Kinase D Activation

“…Specifically, exposure of intact cells to phorbol esters, cell-permeant diacylglycerols, bryostatin, neuropeptide agonists, and growth factors induces rapid PKD phosphorylation and activation that is maintained during cell disruption and immunoprecipitation (13,14,(17)(18)(19). PKD activity recovered from cells stimulated with these agents can be measured by kinase assays in the absence of lipid activators.…”