重组人睾丸精子结合蛋白对人精子蛋白激酶A及蛋白激酶C活性的影响

为探讨人睾丸精子结合蛋白(human testis sperm binding protein,hTSBP)促进人精子获能的分子机制,检测了重组hTSBP对人精子蛋白激酶A(PICA)和蛋白激酶C(PKC)活性的影响.用重组载体pcDNA3.1/myc-His(-)B-tsbp转染真核细胞后,金属亲和层析纯化表达的重组蛋白质His6-TSBP;重组蛋白质处理健康成人精子后,分别提取精子胞浆及胞膜蛋白组分,发现重组蛋白质可以与精子胞膜结合;经放射自显影检测,发现0.1 mg/ml重组蛋白质可以提高人精子中PKA的活性,对精子膜PKC活性无显著影响.     

青藤碱对家兔主动脉血管平滑肌细胞内游离钙浓度及蛋白激酶C的影响

观测青藤碱对培养家兔血管平滑肌细胞内游离钙浓度及正常和缺血缺氧刺激下蛋白激酶C（PKC）活性的影响。方法：Fura-2／AM作Ca^2＋指示剂,检测青藤碱对培养家兔主动脉血管平滑肌细胞静息Ca^2＋浓度及去甲肾上腺素,高K^＋,咖啡因刺激作用下的改变,并与钙拮抗剂维拉帕米进行对照研究;复制血管平滑肌细胞缺血缺氧模型,液闪仪测定PKC活性。结果：青藤碱剂量依赖性抑制高K^＋去极化引起[Ca^2＋]i升高,青藤碱10×10^-6mol.L^-1、3×10^-5mol.L^-1、10^-4mol.L^-1,对NE通过受体介导引起的[Ca^2＋]i增高也有明显抑制。但对静息状态下及咖啡因刺激的血管平滑肌细胞[Ca^2＋]i无明显影响。正常时,青藤碱处理后血管平滑肌细胞胞浆、胞膜PKC活性均升高;缺血缺氧状态下,胞浆PKC活性升高,但胞膜PKC活性降低,青藤碱处理后胞浆PKC活性下降,胞膜PKC活性上升。结论：青藤碱可能抑制血管平滑肌细胞电压依赖性钙通道和受体操纵性钙通道,降低细胞内游离钙水平。调节缺血缺氧条件下血管平滑肌细胞PKC活性。     

ACh对人垂体腺瘤细胞内蛋白激酶C、胞内游离钙及cAMP/cGMP的影响

我们曾发现ACh可明显地抑制垂体腺瘤细胞的增殖代谢,为深入探讨ACh抑制垂体腺瘤细胞增殖作用的机制,观察了ACh作用后垂体腺瘤细胞内蛋白激酶C(PKC)、[Ca^2 ]i及cAMP／cGMP的变化。结果发现：(1)与空白处理组相比,使用PKC的激动剂PMA处理培养的人垂体腺瘤细胞时可使胞浆、胞膜和细胞总PKC活性浓度均升高,但ACh(10μmol/L)作用15min后,胞浆、胞膜和细胞总PKC活性均下降,且此作用可被阿托品阻断;(2)ACh(10μmol/L)作用于单个人垂体腺瘤细胞后,立即使垂体腺瘤细胞[Ca^2 ]i相对水平降低,但此作用可被阿托品阻断;(3)ACh作用于人垂体腺瘤细胞15min后,胞内cAMP水平均明显升高,而cGMP没有改变。该结果为探讨ACh抑制垂体腺瘤细胞增殖的分子机制提供了重要线索,同时提示,ACh对垂体瘤细胞增殖分化的调控作用是细胞内多信息系统相互整合的结果。     

普罗托品对大鼠胸主动脉血管平滑肌细胞内游离钙浓度和蛋白激酶C活性的影响

本实验室先前的研究已证实,普罗托品(protopine,Pro)舒张家兔主动脉的作用,可能与其增加血管平滑肌细胞内cAMP和cGMP水平有关.为了深入探讨Pro的扩血管作用机制,实验采用等张收缩记录大鼠离体血管条张力,利用Fura-2/AM负载的大鼠胸主动脉培养细胞直接测定细胞内游离Ca2+浓度([Ca2+]i),并应用同位素γ-32p-ATP催化活性法测定蛋白激酶C(PKC)活性等方法,分别观察了Pro的相关效应.结果表明,Pro(30和100 μmol/L)明显降低去甲肾上腺素(NA)和高钾所致的动脉条收缩幅度,使二者的量效曲线呈非平行右移,最大反应压低;pD2'值分别为3.7±0.25和3.97±0.15;Pro(50和100μmol/L)对静息状态下[Ca2+]i没有任何影响,但对NA和高钾引起的[Ca2+]i升高均有明显抑制作用;Pro(30和100 μmol/L)对未经NA处理血管条的胞浆和胞膜PKC活性均无明显影响;但在NA预处理的血管条,Pro使NA所升高的胞浆内PKC的活性趋于降低,而明显升高胞膜PKC的活性,对PKC的总活性无明显影响.结果提示,在有NA存在的情况下,Pro似能促使PKC从胞浆向细胞膜转移,其扩血管效应似为其降Ca2+作用、升高cAMP和cGMP的作用及其对PKC影响等几方面的综合结果.     

蛋白激酶C与吗啡耐受

蛋白激酶C(protein kinase C,PKC)属于AGC蛋白激酶家族(即PKA/PKG/PKC激酶家族),在吗啡介导的μ-阿片受体脱敏及吗啡耐受中具有重要作用,因此研究PKC的细胞信号传导机制对吗啡耐受的治疗具有重要的临床意义。本文综述了PKC在吗啡耐受中的作用。     

aFGF对人脐静脉内皮细胞TPK、PKC活性及Ca2+浓度的影响

为了观察酸性成纤维细胞生长因子 ( acidic fibroblast growth factor,a FGF)与人脐静脉内皮细胞 ( human umbilical vein endothelial cell,HUVEC)膜上特异受体结合后引起的细胞内信号转导途径 ,探讨 a FGF导致细胞增殖的机理 ,经 Scatchard曲线分析人脐静脉内皮细胞膜受体性质 .以不同浓度的 a FGF处理人脐静脉内皮细胞 ,利用 [γ- 3 2 P]ATP参入外源性底物的方法测定受体的酪氨酸蛋白激酶 ( tyrosine protein kinase,TPK)及蛋白激酶 C( protein kinase C,PKC)的活性 ;用 Fura-2 /AM为荧光指示剂测定 [Ca2 ]i.结果显示 :Scatchard曲线证明 a FGF与 HUVEC膜受体特异结合呈一条曲线 ,即受体为一种结合位点 ,Kd 为 3.6× 1 0 -10～ 9.6× 1 0 -10 mol/L,每个细胞受体数为2 70 90 .随着 a FGF浓度增加 ,TPK及 PKC活性随之升高 .当 a FGF浓度为 1 .1 2 mg/L时 ,a FGF处理组的 TPK活性是对照组的 3倍 ;膜 PKC活性是对照组 3.4倍 ,胞浆 PKC活性是对照组的 1 .87倍 .胞浆 [Ca2 ]是对照组的 3倍 .结果指出 :该细胞中 a FGF受体具有 TPK活性 .TPK激活后进一步促进蛋白质和酶磷酸化级联反应 ,而使 PKC活性及 [Ca2 ]i 升高 ,即 PKC和 Ca2 为 TPK的下游信号分子 ,进一步促进基因表达增加 ,导致细胞增殖 .     

aFGF对人脐静脉内皮细胞TPK、PKC活性及Ca~(2+)浓度的影响

为了观察酸性成纤维细胞生长因子 ( acidic fibroblast growth factor,a FGF)与人脐静脉内皮细胞 ( human umbilical vein endothelial cell,HUVEC)膜上特异受体结合后引起的细胞内信号转导途径 ,探讨 a FGF导致细胞增殖的机理 ,经 Scatchard曲线分析人脐静脉内皮细胞膜受体性质 .以不同浓度的 a FGF处理人脐静脉内皮细胞 ,利用 [γ- 3 2 P]ATP参入外源性底物的方法测定受体的酪氨酸蛋白激酶 ( tyrosine protein kinase,TPK)及蛋白激酶 C( protein kinase C,PKC)的活性 ;用 Fura-2 /AM为荧光指示剂测定 [Ca2 + ]i.结果显示 :Scatchard曲线证明 a FGF与 HUVEC膜受体特异结合呈一条曲线 ,即受体为一种结合位点 ,Kd 为 3.6× 1 0 -10～ 9.6× 1 0 -10 mol/L,每个细胞受体数为2 70 90 .随着 a FGF浓度增加 ,TPK及 PKC活性随之升高 .当 a FGF浓度为 1 .1 2 mg/L时 ,a FGF处理组的 TPK活性是对照组的 3倍 ;膜 PKC活性是对照组 3.4倍 ,胞浆 PKC活性是对照组的 1 .87倍 .胞浆 [Ca2 + ]是对照组的 3倍 .结果指出 :该细胞中 a FGF受体具有 TPK活性 .TPK激活后进一步促进蛋白质和酶磷酸化级联反应 ,而使 PKC活性及 [Ca2 + ]i 升高 ,即 PKC和 Ca2 + 为 TPK的下游信号分子 ,进一步促进基因表达增加 ,导致细胞增殖 .     

血管活性肠肽对肺表面活性物质结合蛋白A表达的影响

目的:研究血管活性肠肽(VIP)对肺表面活性物质结合蛋白A(SP-A)表达的影响以及VIP调控SP-A表达的细胞内信号转导途径.方法:运用免疫组织化学和RT-PCR技术研究VIP对SP-A表达的影响;并进一步运用受体拮抗、蛋白激酶抑制、反义寡核苷酸阻断等手段探讨VIP促进SP-A表达的信号转导途径.结果:①VIP(10-8mol/L)促进肺泡Ⅱ型细胞(ATⅡ)细胞中的SP-A蛋白表达和提高肺组织SP-AmRNA含量:②VIP受体拮抗剂(10-6mol/L)可取消VIP(10-8mol/L)促进SP-A表达的效应;③蛋白激酶C抑制剂H7(10-5mol/L)和c-fos基因的反义寡核苷酸(9×10 6mol/L)均可阻断VIP促进SP-A表达的作用.结论:VIP通过其受体促进SP-A的表达,PKC及c-fos蛋白在介导VIP促进SP-A表达的细胞内信号转导过程中起重要作用.     

胆囊收缩素8对大鼠大脑皮质细胞钙调素和蛋白激酶C活性的影响

 为阐明中枢神经系统中胆囊收缩素 8(CCK8)受体的信号传递机制 ,以分离的大鼠大脑皮质神经细胞为材料 ,观察了CCK8对细胞内钙调素 (CaM)、3′ ,5′ 环腺苷酸 (cAMP)、蛋白激酶C(PKC)活性的影响 研究结果表明 ,CCK8可刺激大脑皮质细胞CaM、PKC活性的增加 ,并有剂量依赖关系 但CCK8在 10 -12 ～ 10 -6mol L范围内 ,细胞内cAMP含量无显著变化 利用受体亚型L 364,718和L 365,2 60的研究表明 ,两种拮抗剂均可抑制CCK8引起的CaM和PKC活性变化 ,但两者IC50 不同 对于CaM ,CCKB 受体拮抗剂L 365,2 60的IC50 比CCKA 受体拮抗剂L 364,718低 4 0倍 ;而对于PKC ,L 365,2 60的IC50 比L 364,718低 60倍 因此认为 ,CCK8主要是通过CCKB 受体介导了CaM和PKC活性的变化     

蛋氨酸脑啡肽—阿片受体激活免疫细胞抗肿瘤活性的研究进展

蛋氨酸脑啡肽(MENK)是人体中的一种神经递质,其阿片受体广泛存在于免疫细胞表面。MENK与阿片受体相结合,通过调节cAMP-PKA信号通路、Ca2+-钙调蛋白、蛋白激酶C等多种物质的表达,参与到免疫细胞的成熟分化。进一步研究提示,适当剂量的MENK通过激活T细胞、NK细胞、LAK细胞等发挥抗肿瘤的作用。此外,MENK-阿片生长因子受体(OGFr)通路的过表达,直接抑制肿瘤发展与血管生长。     

Dopamine D1 receptor-mediated inhibition of NADPH oxidase activity in human kidney cells occurs via protein kinase A-protein kinase C cross talk

Dopamine cellular signaling via the D(1) receptor (D(1)R) involves both protein kinase A (PKA) and protein kinase C (PKC), but the PKC isoform involved has not been determined. Therefore, we tested the hypothesis that the D(1)R-mediated inhibition of NADPH oxidase activity involves cross talk between PKA and a specific PKC isoform(s). In HEK-293 cells heterologously expressing human D(1)R (HEK-hD(1)), fenoldopam, a D(1)R agonist, and phorbol 12-myristate 13-acetate (PMA), a PKC activator, inhibited oxidase activity in a time- and concentration-dependent manner. The D(1)R-mediated inhibition of oxidase activity (68.1±3.6%) was attenuated by two PKA inhibitors, H89 (10μmol/L; 88±8.1%) and Rp-cAMP (10μmol/L; 97.7±6.7%), and two PKC inhibitors, bisindolylmaleimide I (1μmol/L; 94±6%) and staurosporine (10nmol/L; 93±8%), which by themselves had no effect (n=4-8/group). The inhibitory effect of PMA (1μmol/L) on oxidase activity (73±3.2%) was blocked by H89 (100±7.8%; n=5 or 6/group). The PMA-mediated inhibition of NADPH oxidase activity was accompanied by an increase in PKCθ(S676), an effect that was also blocked by H89. Fenoldopam (1μmol/L) also increased PKCθ(S676) in HEK-hD(1) and human renal proximal tubule (RPT) cells. Knockdown of PKCθ with siRNA in RPT cells prevented the inhibitory effect of fenoldopam on NADPH oxidase activity. Our studies demonstrate for the first time that cross talk between PKA and PKCθ plays an important role in the D(1)R-mediated negative regulation of NADPH oxidase activity in human kidney cells.     

肌醇磷脂激酶(PI4-K)单克隆抗体的制备及其对细胞生长的抑制

制备了抗肌醇磷脂激酶 ( PI4- K)单克隆抗体 ( A6D)并测定了抗原 -抗体反应基本特性及功能 .结果表明 ,单克隆抗体与固相及溶液中肌醇磷脂激酶的亲和常数分别为 7.5× 1 0 6和 6× 1 0 8( mol/L) -1.单抗 1 .9× 1 0 -7mol/L可以抑制从细胞提取液的 PI4- K酶活力 50 % .用 FITC标记单抗在蛋白微球引导下进入细胞内 ,主要富集在细胞质膜区 ,并对 He La细胞和小鼠小脑细胞生长有明显抑制作用 .     

Direct activation of gastric H,K-ATPase by N-terminal protein kinase C phosphorylation. Comparison of the acute regulation mechanisms of H,K-ATPase and Na,K-ATPase

In this study we compared the protein kinase dependent regulation of gastric H,K-ATPase and Na,K-ATPase. The protein kinase A/protein kinase C (PKA/PKC) phosphorylation profile of H,K-ATPase was very similar to the one found in the Na,K-ATPase. PKC phosphorylation was taking place in the N-terminal part of the alpha-subunit with a stoichiometry of approximately 0.6 mol Pi/mole alpha-subunit. PKA phosphorylation was in the C-terminal part and required detergent, as is also found for the Na,K-ATPase. The stoichiometry of PKA-induced phosphorylation was approximately 0.7 mol Pi/mole alpha-subunit. Controlled proteolysis of the N-terminus abolished PKC phosphorylation of native H,K-ATPase. However, after detergent treatment additional C-terminal PKC sites became exposed located at the beginning of the M5M6 hairpin and at the cytoplasmic L89 loop close to the inner face of the plasma membrane. N-terminal PKC phosphorylation of native H,K-ATPase alpha-subunit was found to stimulate the maximal enzyme activity by 40-80% at saturating ATP, depending on pH. Thus, a direct modulation of enzyme activity by PKC phosphorylation could be demonstrated that may be additional to the well-known regulation of acid secretion by recruitment of H,K-ATPase to the apical membranes of the parietal cells. Moreover, a distinct difference in the regulation of H,K-ATPase and Na,K-ATPase is the apparent absence of any small regulatory proteins associated with the H,K-ATPase.     

cAMP对转化细胞中几种基因表达及CREB DNA结合活性的影响

 从癌基因、抑癌基因及转录因子 CREB(c AMP反应序列结合蛋白 )对 CRE DNA序列结合活性的相关性 ,对 db- c AMP处理的小鼠 C3H10 T1 /2转化细胞增殖抑制作用进行了研究 .实验结果表明 ,转化细胞中 PKA(蛋白激酶 A)活性显著低于正常细胞 ,而 PKC(蛋白激酶 C)活性则显著高于正常细胞 .斑点印迹和 Northern印迹分析显示转化细胞中 c- myc和 Ca M(钙调素 )基因表达明显高于正常细胞 ,而 p53基因和 Rb基因表达则明显低于正常细胞 ,这些差别与 C3H10 T1/ 2 转化细胞增殖失控有关 .转化细胞经 db- c AMP(1 mmol/L)处理后 ,细胞增殖受到明显抑制 ,db- c AMP处理0 .5h后 ,转化细胞中 PKA活性便明显增强 ,PKC活性则被显著抑制 ,处理 2 h后 ,c- myc和 Ca M基因表达下降 ,而 p53和 Rb基因表达则增强 ,这些变化与 c AMP抑制 C3H10 T1/ 2 转化细胞增殖有密切联系 .凝胶阻滞电泳分析显示 db- c AMP(1 mmol/L )处理短时间内 ,CREB对 CRE DNA序列无结合活性 ,1 2 h后开始出现较弱的结合活性 ,2 4 h后才明显加强 ,表明在 db- c AMP处理的早期 ,调控区中含有 CRE序列的基因不参与 db- c AMP对细胞增殖抑制的调节 ,即与 CREB磷酸化及其相应的 DNA结合活性无相关性 .     

胞浆Ca~(2+)和某些激酶对NADPH氧化酶激活和肌动蛋白聚合的作用

 为澄清中性粒细胞胞浆 Ca2 +和某些 O-·2 产生相关激酶对 NADPH氧化酶激活和肌动蛋白聚合的作用 ,利用分化为中性粒细胞样的 HL- 60细胞研究了胞浆 Ca2 +螯合剂 BAPTA- AM和激酶抑制剂对这些激酶激活、NADPH氧化酶激活和肌动蛋白聚合的影响 .使用 1 0 μmol/L的 Ca2 +螯合剂 BAPTA- AM去除胞浆 Ca2 +后 ,趋化肽 f MLP诱导的 O-·2 产生明显减少 ,但不影响 f MLP诱导的肌动蛋白聚合 ;8μmol/L的 PKC激酶抑制物 GF1 0 92 0 3x几乎完全抑制 O-·2 产生 ;50 μmol/L的p38激酶抑制物 SB2 0 3580、50 μmol/L的 ERK激酶抑制物 PD0 980 59和 0 .1 μmol/L的 PI3激酶抑制物渥曼青霉素 (Wortmannin)使 f MLP诱导的 O-·2 产生大约减少一半 ;其中 Wortmannin还抑制 f MLP诱导的肌动蛋白聚合 ;f MLP刺激细胞后 ,PI3- K、p38和 ERK激酶迅速激活 ,但这些激酶的激活对 Ca2 +是非必需的 .这些结果说明 Ca2 +依赖途径 (PKC)和 Ca2 +非依赖途径 (PI3- K、p38和ERK)对 NADPH氧化酶激活都起着重要作用 ,而 Ca2 +非依赖途径中的 PI3- K激酶还参与中性粒细胞样 HL- 60细胞的肌动蛋白聚合 .     

Effect of parathyroid hormone on rat renal cAMP-dependent protein kinase and protein kinase C activity measured using synthetic peptide substrates

The actions of parathyroid hormone (PTH) on the renal cortex are thought to be mediated primarily by cAMP-dependent protein kinase (PKA) with some suggestion of a role for protein kinase C (PKC). However, present methods for assaying PKA and PKC in subcellular fractions are insensitive and require large amounts of protein. Recently, a sensitive method for measuring the activity of protein kinases has been reported. This method uses synthetic peptides as substrates and a tandem chromatographic procedure for isolating the phosphorylated peptides. We have adapted this method to study the effect of PTH on PKA and PKC activity using thin slices of rat renal cortex. PTH (250 nM) stimulated cytosolic PKA activity four- to fivefold within 30 s, and PKA activity was sustained for at least 5 min. PTH also rapidly stimulated PKC activity in the membrane fraction and decreased PKC activity in the cytosol. These changes were maximal at 30 s, but unlike changes in PKA, they declined rapidly thereafter. PTH significantly activated PKC only at concentrations of 10 nM or greater. This study demonstrates that PTH does activate PKC in renal tissue, although the duration of activation is much less than for PKA. It also demonstrates that a combination of synthetic peptides with tandem chromatography can be used as a sensitive assay procedure for protein kinase activity in biological samples.     

Mechanism of CNP-mediated DG-PKC and IP4 signaling pathway in diabetic rats with gastric motility disorder

In the precedent research conducted by the same team, it concluded that the activities in C-type natriuretic peptide (CNP)/cyclic guanosine monophosphate (cGMP)/cyclic adenosine monophosphate (cAMP)/β-type phospholipase C (PLCβ) pathways of rat antral smooth muscle were changed due to diabetes, which was the key pathogenetic mechanism for diabetic gastric dysmotility. As the follow-on step, this study was designed to probe into the downstream signaling pathway of CNP/PLCβ. The results showed that level of α-type protein kinase C (PKCα),cell membrane to cytoplasm ratio of PKCα, cell membrane to cytoplasmic ratio of βI-type protein kinase C (PKCβI) and level of Phosphor-PKCα (P-PKCα) were significantly reduced in diabetes rat antral smooth muscle samples. The content of tetraphosphate inositol (IP4) in gastric antral smooth muscle of diabetic rats reduced, and the content of diacyl-glycerol (DG) was unchanged. CNP significantly decreased the content of IP4 and DG, this effect was more obvious in diabetic rats. Subsequent to the addition of protein kinase A (PKA) blocker N-[2- (p-Bromocin-namylamino)ethyl]-5 -isoquinolinesulfonamide dihydrochloride (H-89) before CNP treatment, the inhibitory effect of CNP was reduced; subsequent to the addition of protein kinase G (PKG) blocker KT5823 before CNP treatment, the inhibitory effect of CNP was also reduced. With the addition of the combination of H-89 and KT5823 before CNP treatment, the inhibition by CNP could be offset. These results were concluded that CNP inhibited the activity of PKC family in rat smooth muscle and reduced the levels of IP4 and DG through the PKG/PKA-PLCβ pathways, causing inhibited muscular contractions, which may be a key pathogenetic factor for diabetic gastroparesis.

     

Interaction of protein kinase C and cAMP-dependent pathways in the phosphorylation of the Na,K-ATPase

To test the hypothesis that there is cross-talk between the protein kinase C (PKC) and protein kinase A (PKA) pathways in the regulation of the Na,K-ATPase, we measured its phosphorylation in mammalian cell cultures. Phosphorylation of the PKC site, Ser-18, appeared to be due to the activation of the alpha isoform of the kinase. In NRK-52E and L6 cells, this phosphorylation was reduced by prior activation of a cAMP-dependent signaling pathway with forskolin. In principle this would be consistent with direct interaction between the two phosphorylation sites, but further investigation suggested a more indirect mechanism. First, phosphorylation of Ser-938, the PKA site, could not be detected despite the presence of active PKA. Second, there was a major reduction in the phosphorylation of unrelated phosphoproteins as a consequence of elevation of cAMP, suggesting generalized reduction of kinase activity or activation of phosphatase activity. In NRK-52E and L6, phosphorylation of the Na, K-ATPase at Ser-18 paralleled this global change. In C6 cells, in contrast, there was no cAMP effect on Na,K-ATPase phosphorylation at Ser-18 and no global cAMP effect on other phosphoproteins. The cross-talk is evidently mediated by events occurring at the cellular level.     

Comparative effects of polymyxin B, phorbol ester and bryostatin on protein phosphorylation, protein kinase C translocation, phospholipid metabolism and differentiation of HL60 cells

The effects of protein kinase C (PKC) inhibitor polymyxin B (PMB) and PKC activators 12-O-tetradecanoylphorbol-13-acetate (TPA) and bryostatin on intact HL60 cells were examined. It was found that each of the three agents exhibited similar effects on phosphorylation of certain endogenous proteins, PKC translocation from cytoplasm to plasma membrane and formation of CDP-choline. TPA, however, was the only agent that stimulated phosphatidylcholine formation. Differentiation of HL60 cells was potently induced by TPA; in comparison bryostatin was a relatively weaker inducer and PMB was without effect. The data indicated that the effects of the PKC inhibitor PMB on intact cells could not be predicted by its in vitro activity, and that certain TPA-dependent but PKC-independent reactions might be crucial in HL60 cell differentiation.