Differential Activation Of Protein Kinase Research Articles

IL-32-PAR2 axis is an innate immunity sensor providing alternative signaling for LPS-TRIF axis

Interleukin (IL)-32 is known to exert adujvant effects on innate immune response, however, receptors and downstream signaling pathways remain to be clarified. Here we found that IL-32γ upregulated serine protease activity of proteinase-3 (PR3), in turn triggering protease-activated receptor 2 (PAR2) signaling. Interestingly, silencing of PR3 or PAR2 using siRNA markedly diminished IL-32γ-induced TNFα and IFN-β mRNA expression. IL-32γ-PAR2 axis utilized TRIF and Ras-Raf-1 pathways. On stimulation with lipopolysaccharide (LPS), differential activation of protein kinase C isoforms modulated the balance between LPS-TLR4-TRIF and IL-32-PAR2-TRIF axes, because LPS was a strong inducer of IL-32γ. IL-32-PAR2-TRIF axis might serve not only as an extracellular sensor of bacterial and autologous proteases, but also as a modulator of innate and adaptive immunity during infection.

Editorial: Molecular Endocrinology Articles in the Spotlight for April 2013

We are pleased to feature the following articles in this month’s issue of Molecular Endocrinology. Interestingly, the 3 articles provide novel insights into various hypothalamic pathways that regulate important aspects of reproduction and metabolism. In “GnRH Pulse Frequency-Dependent Stimulation of FSH Transcription Is Mediated via Activation of PKA and CREB,” Thompson et al provide a mechanism of GnRH pulse frequency-dependent activation of FSH involving differential activation of protein kinase A (PKA) and subsequent differential levels of cAMP-responsive element-binding protein (CREB) phosphorylation. LH gene expression is unaffected by this pathway. Furthermore, GnRH preferentially stimulates CREB phosphorylation at slow pulse frequencies, whereas PKA (the main regulator of CREB phosphorylation in the murine LT2 gonadotrope cell model) activity increases to a greater extent at slow GnRH pulse frequencies, correlating with the increase in CREB phosphorylation. Because pulsatile GnRHregulatestheexpressionandsecretionofbothFSH and LH, this work identifies the intracellular mechanism downstream of GnRH receptor activation responsible for triggering the diverse reproductive consequences of the individual gonadotropins. “Nuclear Receptor LRH-1 Induces the Reproductive Neuropeptide Kisspeptin in the Hypothalamus” by Atkin et al (freely available at http://mend.endojournals.org/) provides novel insights into the molecular mechanism controlling kisspeptin’s differential secretion. The authors show that mice lacking LRH-1 in kisspeptin neurons have reduced plasma FSH levels and corresponding defects in follicle maturation and ovulation in the ovary. These knockout mice have a prolonged estrous cycle and deliver fewer pups per litter. On a molecular level, these findings help explain why kisspeptin is differentially regulatedindistinctregionsofthehypothalamus,thearcuate and anteroventral periventricular nuclei, and have revealed an important role for LRH-1 in regulating the female reproductive axis. Groba et al’s “Hypothyroidism Compromises Hypothalamic Leptin Signaling in Mice” reveals that leptin signaling in hypothalamic arcuate neurons is compromised under hypothyroid conditions. Activation of the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathways by leptin is reduced in hypothyroid obese (ie, ob/ob) mice as evidenced by reduced phopho-STAT3 immunoreactivity and decreased SOCS3 expression. A diminished reduction in body weight and food intake in hypothyroid ob/ob mice was observed after leptin treatment. Importantly, hypothyroidism is associated with elevated transcript levels of shorter leptin receptor isoforms. Consequently, up-regulation of the shorter isoforms of the leptin receptor may represent a major mechanism by which hypothyroidism influences the leptin signaling pathway in hypothyroid mice.

Editorial: Molecular Endocrinology Articles in the Spotlight for April 2012

We are pleased to feature the following articles in this month’s issue of Molecular Endocrinology. Interestingly, the 3 articles provide novel insights into various hypothalamic pathways that regulate important aspects of reproduction and metabolism. In “GnRH Pulse Frequency-Dependent Stimulation of FSH Transcription Is Mediated via Activation of PKA and CREB,” Thompson et al provide a mechanism of GnRH pulse frequency-dependent activation of FSH involving differential activation of protein kinase A (PKA) and subsequent differential levels of cAMP-responsive element-binding protein (CREB) phosphorylation. LH gene expression is unaffected by this pathway. Furthermore, GnRH preferentially stimulates CREB phosphorylation at slow pulse frequencies, whereas PKA (the main regulator of CREB phosphorylation in the murine LT2 gonadotrope cell model) activity increases to a greater extent at slow GnRH pulse frequencies, correlating with the increase in CREB phosphorylation. Because pulsatile GnRHregulatestheexpressionandsecretionofbothFSH and LH, this work identifies the intracellular mechanism downstream of GnRH receptor activation responsible for triggering the diverse reproductive consequences of the individual gonadotropins. “Nuclear Receptor LRH-1 Induces the Reproductive Neuropeptide Kisspeptin in the Hypothalamus” by Atkin et al (freely available at http://mend.endojournals.org/) provides novel insights into the molecular mechanism controlling kisspeptin’s differential secretion. The authors show that mice lacking LRH-1 in kisspeptin neurons have reduced plasma FSH levels and corresponding defects in follicle maturation and ovulation in the ovary. These knockout mice have a prolonged estrous cycle and deliver fewer pups per litter. On a molecular level, these findings help explain why kisspeptin is differentially regulatedindistinctregionsofthehypothalamus,thearcuate and anteroventral periventricular nuclei, and have revealed an important role for LRH-1 in regulating the female reproductive axis. Groba et al’s “Hypothyroidism Compromises Hypothalamic Leptin Signaling in Mice” reveals that leptin signaling in hypothalamic arcuate neurons is compromised under hypothyroid conditions. Activation of the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathways by leptin is reduced in hypothyroid obese (ie, ob/ob) mice as evidenced by reduced phopho-STAT3 immunoreactivity and decreased SOCS3 expression. A diminished reduction in body weight and food intake in hypothyroid ob/ob mice was observed after leptin treatment. Importantly, hypothyroidism is associated with elevated transcript levels of shorter leptin receptor isoforms. Consequently, up-regulation of the shorter isoforms of the leptin receptor may represent a major mechanism by which hypothyroidism influences the leptin signaling pathway in hypothyroid mice.

Differential activation of protein kinase C isoforms following chemical ischemia in rat cerebral cortex slices

The aim of the current study was to characterize the effects of chemical ischemia and reperfusion at the transductional level in the brain. Protein kinase C isoforms (α, β 1, β 2, γ, δ and ɛ) total levels and their distribution in the particulate and cytosolic compartments were investigated in superfused rat cerebral cortex slices: (i) under control conditions; (ii) immediately after a 5-min treatment with 10 mM NaN 3, combined with 2 mM 2-deoxyglucose (chemical ischemia); (iii) 1 h after chemical ischemia (reperfusion). In control samples, all the PKC isoforms were detected; immediately after chemical ischemia, PKC β 1, δ and ɛ isoforms total levels (cytosol + particulate) were increased by 2.9, 2.7 and 9.9 times, respectively, while α isoform was slightly reduced and γ isoform was no longer detectable. After reperfusion, the changes displayed by α, β 1, γ, δ and ɛ were maintained and even potentiated, moreover, an increase in β 2 (by 41 ± 12%) total levels became significant. Chemical ischemia-induced a significant translocation to the particulate compartment of PKC α isoform, which following reperfusion was found only in the cytosol. PKC β 1 and δ isoforms particulate levels were significantly higher both in ischemic and in reperfused samples than in the controls. Conversely, following reperfusion, PKC β 2 and ɛ isoforms displayed a reduction in their particulate to total level ratios. The intracellular calcium chelator, 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid, 1 mM, but not the N-methyl- d-asparate receptor antagonist, MK-801, 1 μM, prevented the translocation of β 1 isoform observed during ischemia. Both drugs were effective in counteracting reperfusion-induced changes in β 2 and ɛ isoforms, suggesting the involvement of glutamate-induced calcium overload. These findings demonstrate that: (i) PKC isoforms participate differently in neurotoxicity/neuroprotection events; (ii) the changes observed following chemical ischemia are pharmacologically modulable; (iii) the protocol of in vitro chemical ischemia is suitable for drug screening.

Activation of nuclear transcription factor-kappa B is associated with the induction of inhibitory kappa B kinase-beta and involves differential activation of protein kinase C and protein tyrosine kinases during fatal murine cerebral malaria

The levels of nuclear transcription factor-kappa B (NF-κB) subunits p65 and p50 and its associated kinase, inhibitory kappa B kinase (IKK) alpha and beta were monitored in cytosolic and nuclear fraction of mice cerebral cortex and cerebellum using an experimental model of fatal murine cerebral malaria (FMCM). Since protein kinase C (PKC) and protein tyrosine kinases (PTK) are known to collaborately regulate the NF-κB activation, we also studied the activity of these two kinases in cytosol and membrane fraction. In parallel, the levels of two PKC isoforms (alpha and delta) and tyrosine phosphorylated proteins were monitored to correlate the observed changes in the activity. Our results underscore the involvement of IKK-beta as an essential mediator of NF-κB activation as evinced by the nuclear translocation of p65 and p50 during CM pathology. Additional findings confirm altered activity and levels of PKC and enhanced activation of PTK and tyrosine phosphorylation of proteins during CM pathology. These signaling intricacies involving an interplay between rel family (NF-κB) of transcription factors, PKC and PTK may serve as an important cue in understanding the possible continuation of the post receptor signaling events associated with tumor necrosis factor-alpha induction during FMCM pathology.

Differential Activation of Protein Kinase C between Ischemic and Pharmacological Preconditioning in the Rabbit Heart.

The objective of the present study was to investigate the differential activation of protein kinase C between ischemic (IPC) and pharmacological preconditioning (PPC) in the rabbit heart. Control, IPC, diazoxide (Diaz), and chelerythrine (Chel)+IPC groups underwent prolonged coronary artery occlusion (CAO) for 30 minutes followed by 180 minutes' reperfusion (protocol I). In protocol II, sham, IPC-only, Diaz-only, and Chel+IPC-only groups did not undergo prolonged CAO. IPC was induced with 4 cycles of 5-min regional ischemia and 10-min reperfusion before prolonged CAO. Diaz (5 mg/kg) was administered 30 min before prolonged CAO. Chel (5 mg/kg) was administered 5 min before the IPC procedure. Infarct size was determined by tetrazolium staining. Assessment of protein kinase C (PKC) isoforms from a left ventricular (LV) sample was conducted by western blotting. Apoptosis in situ was determined by TUNEL assay. The infarction area in the IPC (11.6 +/- 1.0%) and Diaz (19.5 +/- 3.8%) groups was reduced significantly (p< 0.01, p< 0.05) relative to the control group (40.0 +/- 3.8%). The reduction by IPC was abolished by pretreatment with Chel. Apoptosis was significantly decreased (p< 0.01) in the IPC and diazoxide groups compared with the control and Chel+IPC groups (control: 4.78 +/- 0.56% vs. IPC: 2.00 +/- 0.38% vs. Diaz: 2.20 +/- 0.32% vs. Chel+IPC: 4.32 +/- 0.41%) and DNA laddering was attenuated in the IPC and Diaz groups. Membrane PKC-epsilon levels in the IPC and Diaz groups increased significantly relative to the control and Chel+IPC groups. Membrane PKC-epsilon levels in the IPC-only group showed greater increases than the Diaz-only and Chel+IPC-only groups. These findings suggest that whereas PPC suppresses apoptosis when diazoxide opens mitochondrial K(ATP) channels and then activates PKC-epsilon through ischemia-reperfusion, IPC activates PKC-epsilon in the particulate fraction prior to continuous ischemia-reperfusion. We concluded that the difference between IPC and PPC appears to consist in the difference in the timing of PKC-epsilon activation, though both IPC and PPC provide the cardioprotection in ischemia-reperfusion injury.

Differential Activation of Protein Kinase B and p70S6K by Glucose and Insulin-like Growth Factor 1 in Pancreatic β-Cells (INS-1)

It has been shown that IGF-1-induced pancreatic beta-cell proliferation is glucose-dependent; however, the mechanisms responsible for this glucose dependence are not known. Adenoviral mediated expression of constitutively active phosphatidylinositol 3-kinase (PI3K) in the pancreatic beta-cells, INS-1, suggested that PI3K was not necessary for glucose-induced beta-cell proliferation but was required for IGF-1-induced mitogenesis. Examination of the signaling components downstream of PI3K, 3-phosphoinositide-dependent kinase 1, protein kinase B (PKB), glycogen synthase kinase-3, and p70-kDa-S6-kinase (p70(S6K)), suggested that a major part of glucose-dependent beta-cell proliferation requires activation of mammalian target of rapamycin/p70(S6K), independent of phosphoinositide-dependent kinase 1/PKB activation. Adenoviral expression of the kinase-dead form of PKB in INS-1 cells decreased IGF-1-induced beta-cell proliferation. However, a surprisingly similar decrease was also observed in adenoviral wild type and constitutively active PKB-infected cells. Upon analysis of extracellular signal-regulated protein kinase 1 and 2 (ERK1/ERK2), an increase in ERK1/ERK2 phosphorylation activation by glucose and IGF-1 was observed in kinase-dead PKB-infected cells, but this phosphorylation activation was inhibited in the constitutively active PKB-infected cells. Hence, there is a requirement for the activation of both ERK1/ERK2 and mammalian target of rapamycin/p70(S6K) signal transduction pathways for a full commitment to glucose-induced pancreatic beta-cell mitogenesis. However, for IGF-1-induced activation, these pathways must be carefully balanced, because chronic activation of one (PI3K/PKB) can lead to dampening of the other (ERK1/2), reducing the mitogenic response.

Differential Activation of Protein Kinase A in Various Regions of Myocardium during Sepsis

Changes in the activities of protein kinase A (PKA) (cAMP-dependent protein kinase) in various regions of rat myocardium during different cardiodynamic phases of sepsis were studied in an attempt to understand the pathophysiology of cardiac dysfunction during sepsis. Sepsis was induced by cecal ligation and puncture (CLP). Experiments were divided into three groups: control, early sepsis, and late sepsis. Early and late sepsis refers to those animals sacrificed at 9 and 18 hr, respectively, after CLP. Cardiac PKA was extracted and partially purified by acid precipitation, ammonium sulfate fractionation, and DEAE–cellulose chromatography. PKA was eluted from DEAE–cellulose column with a linear NaCl gradient. Two types of PKA, Type I (eluted at low ionic strength) and Type II (eluted at high ionic strength), were collected, and their activities were determined based on the rate of incorporation of [γ-32P]ATP into histone. Under physiological conditions, Type I PKA activities were unevenly distributed (left atrium > right atrium > pacemaker region > left ventricle > right ventricle > ventricular septum) while Type II PKA activities were evenly distributed among different regions of myocardium. During early sepsis, Type I PKA activities remained unchanged while Type II PKA activities were activated by 32 and 70% in right atrium and pacemaker regions, respectively. During late sepsis, Type I PKA activities were stimulated by 228% in ventricular septum while Type II PKA activities were not affected. These data demonstrate that different PKA activities exist in various regions of the myocardium and that PKA activities were preferentially activated in certain areas during the progression of sepsis. Since PKA plays an important role in the regulation of myocardial function and metabolism, the activation of PKA in different regions of myocardial during different stages of sepsis may contribute to the altered cardiac function during the progression of sepsis.

Differential Activation of Protein Kinase C δ and ε Gene Expression by Gonadotropin-releasing Hormone in αT3-1 Cells: AUTOREGULATION BY PROTEIN KINASE C

The effect of gonadotropin-releasing hormone (GnRH) upon protein kinase C (PKC) delta and PKCepsilon gene expression was investigated in the gonadotroph-derived alphaT3-1 cell line. Stimulation of the cells with a stable analog [D-Trp6]GnRH (GnRH-A) resulted in a rapid elevation of PKCepsilon mRNA levels (1 h), while PKCdelta mRNA levels were elevated only after 24 h of incubation. The rapid elevation of PKCepsilon mRNA by GnRH-A was blocked by pretreatment with a GnRH antagonist or actinomycin D. The PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA), but not the Ca2+ ionophore ionomycin, mimicked the rapid effect of GnRH-A upon PKCepsilon mRNA elevation. Additionally, the rapid stimulatory effect of GnRH-A was blocked by the selective PKC inhibitor GF109203X, by TPA-mediated down-regulation of endogenous PKC, or by Ca2+ removal. Interestingly, serum-starvation (24 h) advanced the stimulation of PKCdelta mRNA levels by GnRH-A and the effect could be detected at 1 h of incubation. The rapid effect of GnRH-A upon PKCdelta mRNA levels in serum-starved cells was mimicked by TPA, but not by ionomycin, and was abolished by down-regulation of PKC or by Ca2+ removal. Preactivation of alphaT3-1 cells with GnRH-A for 1 h followed by removal of ligand and serum resulted in elevation of PKCdelta mRNA levels after 24 h of incubation. Western blot analysis revealed that GnRH-A and TPA stimulated (within 5 min) the activation and some degradation of PKCdelta and PKCepsilon. We conclude that Ca2+ and PKC are involved in GnRH-A elevation of PKCdelta and PKCepsilon mRNA levels, with Ca2+ being necessary but not sufficient, while PKC is both necessary and sufficient to mediate the GnRH-A response. A serum factor masks PKCdelta but not PKCepsilon mRNA elevation by GnRH-A, and its removal exposes preactivation of PKCdelta mRNA by GnRH-A which can be memorized for 24 h. PKCdelta and PKCepsilon gene expression evoked by GnRH-A is autoregulated by PKC, and both isotypes might participate in the neurohormone action.

Differential Activation of Protein Kinase C Isozymes by Phorbol Ester and Collagen in Human Skin Microvascular Endothelial Cells

Human dermal microvascular endothelial cells participate in activities including inflammation, wound healing, and angiogenesis (neovascularization). Two stages of angiogenesis can be mimicked in vitro by two models of cultured foreskin human dermal microvascular endothelial cells: the differentiation of epithelioid endothelial cells to spindle-shaped mesenchymal-like cells induced by phorbol ester treatment; and the formation of vascular channels induced by exposing the luminal surface of endothelial cell monolayers to type I collagen gels. The mechanisms underlying these two processes, however, are largely unknown. Protein kinase C isozymes, which are activated by phorbol esters, are important mediators in the angiogenic process. In the current work, we identified the protein kinase C isozymes present in human dermal microvascular endothelial cells and determined which of the isozymes are activated in response to phorbol ester or to collagen treatments. Using western blot analysis, we found that microvascular endothelial cells contain at least six protein kinase C isozymes (alpha, beta, delta, epsilon, zeta, eta). Immunocytochemical studies demonstrated that the isozymes are located in distinct cellular compartments and that following treatment with phorbol 12-myristate 13-acetate or with a collagen gel overlay, most isozymes (protein kinase C alpha, beta1, betaII, delta, epsilon, eta) translocated to different parts of the cell. Moreover, for two of these isozymes (betaII and eta), the localization differs after phorbol 12-myristate 13-acetate treatment as compared with collagen treatment. These results demonstrate that agents that mimic two stages in the angiogenic process in vitro initiate diverse changes in the subcellular localization of specific protein kinase C isozymes and suggest a role for different isozymes in this process.

Differential activation of protein kinase C isoforms by endothelin-1 and phenylephrine and subsequent stimulation of p42 and p44 mitogen-activated protein kinases in ventricular myocytes cultured from neonatal rat hearts

The translocation of protein kinase C (PKC) isoforms PKC-alpha, PKC-delta, PKC-epsilon, and PKC-zeta from soluble to particulate fractions was studied in ventricular cardiomyocytes cultured from neonatal rats. Endothelin-1 (ET-1) caused a rapid ETA receptor-mediated translocation of PKC-delta and PKC-epsilon (complete in 0.5-1 min). By 3-5 min, both isoforms were returning to the soluble fraction, but a greater proportion of PKC-epsilon remained associated with the particulate fraction. The EC50 of translocation for PKC-delta was 11-15 nM ET-1 whereas that for PKC-epsilon was 1.4-1.7 nM. Phenylephrine caused a rapid translocation of PKC-epsilon (EC50 = 0.9 microM) but the proportion lost from the soluble fraction was less than with ET-1. Translocation of PKC-delta was barely detectable with phenylephrine. Neither agonist caused any consistent translocation of PKC-alpha or PKC-zeta. Activation of p42 and p44 mitogen-activated protein kinase (MAPK) by ET-1 or phenylephrine followed more slowly (complete in 3-5 min). Phosphorylation of p42-MAPK occurred simultaneously with its activation. The proportion of the total p42-MAPK pool phosphorylated in response to ET-1 (50%) was greater than with phenylephrine (20%). In addition to activation of MAPK, an unidentified p85 protein kinase was activated by ET-1 in the soluble fraction whereas an unidentified p58 protein kinase was activated in the particulate fraction.

Differential activation of protein kinase C isozymes by short chain phosphatidylserines and phosphatidylcholines.

To investigate the importance of the physical state of phospholipids for activation of protein kinase C, we have used short chain phospholipids, which, depending on their concentration, can exist as either monomers or micelles. We previously reported that short chain phosphatidylcholines (PC) can activate protein kinase C at concentrations that correlate with the critical micelle concentration of the activating lipid (Walker, J. M., and Sando, J. J. (1988) J. Biol. Chem. 263, 4537-4540). We have now expanded this work to short chain phosphatidylserine (PS) systems in order to examine the role of Ca2(+)-phospholipid interactions in the activation process. Short chain PS were synthesized from corresponding PC and purified by reverse-phase high pressure liquid chromatography. Use of the short chain system has revealed significant differences in the activation of type II and type III protein kinase C isozymes. The type II isozyme required Ca2+ in the presence of long chain PS vesicles; in the presence of the short chain phospholipid micelles (PC or PS), most of the activity was Ca2+ independent. Addition of diacylglycerol caused a small increase in type II activity in all phospholipid systems. In contrast, type III protein kinase C was Ca(+)-dependent in all of the lipid systems. The concentration of Ca2+ required to activate type III protein kinase C was independent of the phospholipid type despite large differences in the ability of these lipids to bind Ca2+. This isozyme required diacylglycerol only in the PC micelle system or with vesicles composed of long chain saturated PS. The presence of short chain PS micelles or long chain PS with unsaturated fatty acyl chains rendered this Ca2(+)-dependent protein kinase C virtually diacylglycerol independent. These results are consistent with a model in which type II protein kinase C requires Ca2+ primarily for membrane association, a requirement which is bypassed with the micelle system, whereas type III protein kinase C has an additional Ca2+ requirement for activity that does not involve Ca2(+)-phospholipid interactions.

Differential activation of protein kinase C alpha is associated with arachidonate release in Madin-Darby canine kidney cells.

The heterogeneity of the protein kinase C (PKC) gene family strongly suggests that different isoforms may have distinct functions in mediating signal transduction. However, there is very little direct evidence for this. PKC has been implicated in arachidonate (AA) release in many cell types. We sought to investigate whether bradykinin- and phorbol ester-stimulated AA release in Madin-Darby canine kidney (MDCK) cells was correlated with differential activation of PKC isoforms. Using phorbol esters to (i) activate the enzyme and (ii) to down-regulate it, we report that differential activation (translocation) of PKC alpha is associated with AA release in MDCK cells and that specific down-regulation of PKC alpha is associated with a loss of AA release in response to stimulation with dioctanoylglycerol and phorbol ester. We also demonstrate that bradykinin-stimulated AA release was associated with differential activation of PKC alpha and was inhibited in PKC alpha down-regulated cells. Thus, we conclude that the PKC alpha isoform is likely to be responsible for mediating AA release in these cells.

Phosphorylation of lamin B at the nuclear membrane by activated protein kinase C.

Both bryostatin 1 and 4 beta-phorbol 12,13-dibutyrate (PBt2) activate Ca2+- and phospholipid-dependent protein kinase (protein kinase C) at the plasma membrane in HL-60 cells (Kraft, A. S., Baker, V. V., and May, W. S. (1987) Oncogene 1, 91-100). However, whereas PBt2 causes HL-60 cells to cease dividing and differentiate, bryostatin 1 antagonizes this effect and allows cells to continue proliferating. To test whether these divergent effects could be due to the differential activation of protein kinase C at the nuclear level, the phosphorylation of nuclear envelope polypeptides was evaluated in cells treated with either bryostatin 1 or PBt2. Bryostatin 1, either alone or in combination with PBt2, but not PBt2 alone, mediates rapid and specific phosphorylation of several nuclear envelope polypeptides. A major target for bryostatin-induced phosphorylation is the major nuclear envelope polypeptide lamin B (Mr = 67,000, pI 6.0). In vitro studies combining purified protein kinase C and HL-60 cell nuclear envelopes demonstrate that bryostatin activates protein kinase C to phosphorylate lamin B, whereas PBt2 does so only weakly, suggesting selective activation of this enzyme toward this substrate. Comparative phosphopeptide and phosphoamino acid analyses demonstrate that bryostatin induces phosphorylation of identical serine sites on lamin B both in whole cells and in vitro. Treatment of whole cells with bryostatin, but not PBt2, leads to specific translocation of activated protein kinase C to the nuclear envelope. Since phosphorylation of lamin B is known to be involved in nuclear lamina depolymerization at the time of mitosis, it is possible that bryostatin-activated protein kinase C activity is involved in this process. Finally, specific activation of protein kinase C at the nuclear membrane could explain, at least in part, the divergent effects of bryostatin 1 and PBt2 on HL-60 cell growth.