植物中鈣依賴蛋白激酶(CDPK)的研究進展

武志剛，武舒佳，王迎春，鄭琳琳

(內(nèi)蒙古大學生命科學學院，內(nèi)蒙古 呼和浩特 010021)

植物中存在著復(fù)雜的信號轉(zhuǎn)導(dǎo)網(wǎng)絡(luò)，用于調(diào)控植物的新陳代謝，以應(yīng)對不斷變化的生存環(huán)境。Ca2+作為重要的第二信使，經(jīng)Ca2+受體蛋白、鈣調(diào)素(CaM)和鈣依賴蛋白激酶(calcium-dependent protein kinase，CDPK)等協(xié)同作用[1]，將鈣信號向下游傳遞并級聯(lián)放大，促進響應(yīng)蛋白的產(chǎn)生，從而調(diào)控植物的生長發(fā)育以及免疫應(yīng)答和脅迫響應(yīng)。

CDPK是目前植物中研究較多、了解較為清楚的一類絲氨酸/蘇氨酸蛋白激酶，在植物發(fā)育及其對生物和非生物脅迫信號轉(zhuǎn)導(dǎo)的應(yīng)答中執(zhí)行不同的生物學功能[2-4]。基因組數(shù)據(jù)分析顯示，在擬南芥(Arabidopsisthaliana)、水稻(Oryzasativa)、小麥(Triticumaestivum)、玉米(Zeamays)及楊樹(Populustomentosa)中分別含有34、31、20、40、20個CDPK基因[5-6]。后續(xù)研究發(fā)現(xiàn)CDPK同樣存在于綠藻類、卵菌和纖毛蟲、頂覆蟲等部分原生動物中。CDPK在不同組織中的表達、亞細胞定位、作用底物以及與不同蛋白體形成復(fù)合物等方面的差異，決定了該家族成員功能的差異，因此，本研究擬從這些方面對CDPKs進行全面的概述。

1 CDPK的結(jié)構(gòu)特征及調(diào)控機制

CDPK首次發(fā)現(xiàn)于豌豆(Pisumsativum)中，第1次從大豆(Glycinemax)中得到純化和鑒定。與其他鈣結(jié)合蛋白不同，CDPK是單肽鏈結(jié)構(gòu)。如圖1所示，CDPK具有4個典型的結(jié)構(gòu)域，從N端到C端依次為N-末端的可變域、催化域(激酶域)、連接域(自抑制域)和調(diào)控域(類鈣調(diào)素域/CaM-LD)。

圖1 CDPK的結(jié)構(gòu)特征Fig.1 Structural features of CDPK

N-末端可變域序列相似性低，保守性差[7]。多數(shù) CDPK 在N-端可變域含有與膜定位相關(guān)的豆蔻?；稽c(第2位的甘氨酸殘基)以及棕櫚?；稽c(第4或第5位的半胱氨酸殘基)[8]，參與CDPK的膜結(jié)合過程。此外，N-末端可變域還在調(diào)節(jié)底物特異性過程中扮演關(guān)鍵的角色[9-10]；催化域也稱激酶域，具有典型的Ser/Thr蛋白激酶的保守序列，同源性較高[11]，該區(qū)域突變會導(dǎo)致催化活性喪失；連接域高度保守，Liese等[7]的研究表明，連接域功能上相當于一種假底物，與催化域結(jié)合保證CDPK處于非活躍狀態(tài)，因此也叫自抑制域；C-末端調(diào)控域上具有類似鈣調(diào)素的結(jié)構(gòu)，因此CDPK的激活依賴于鈣離子而不依賴于鈣調(diào)素。調(diào)控域包含4個可與鈣離子結(jié)合的EF-手性結(jié)構(gòu)，是Ca2+和CDPK的結(jié)合位點[12]。根據(jù)對Ca2+的親和性不同將其分為兩個球形結(jié)構(gòu)，分別為N-lobe和C-lobe[13]，N-lobe對Ca2+親和性低，C-lobe對Ca2+親和性高，當Ca2+濃度低時與C-lobe結(jié)合，當Ca2+濃度高時與N-lobe結(jié)合，與連接域相互作用，導(dǎo)致調(diào)控域發(fā)生構(gòu)象變化，解除自抑制。

2 CDPK的分布與亞細胞定位

2.1 CDPK的表達特性及其在植株內(nèi)的分布

CDPK在植株中分布極其廣泛，器官水平上，在植株的根、莖、葉、花、果實及種子中均能檢測到基因的表達；細胞水平上，CDPK基因普遍存在于分生組織細胞、木質(zhì)部細胞、保衛(wèi)細胞、花粉母細胞及胚胎細胞中。CDPK的家族成員的表達特點不盡相同，有些可以在植物的大多數(shù)器官或組織中表達，如煙草(Nicotianatabacum)NtCPK1在葉片中不表達，在根、莖和花中均有表達[14]；擬南芥的AtCPK12在根、莖、葉、花和成熟果莢等組織中均有表達，但在干種子中不表達[15]；油菜(Brassicacampestris)BnCDPK1 在根、莖、葉、花和種子中都有表達，而有些卻只在特定的組織表達，如AtCPK16、AtCPK17、AtCPK25、AtCPK34只在花粉管中表達[16]；此外，CDPK在不同器官中表達豐度也存在差異，油菜BnCDPK1在葉片中表達量最高，其次為莖、根和種子，在花中的表達量最低[17]。

2.2 CDPK的亞細胞定位及其影響因素

通過亞細胞定位研究表明，CDPK可定位于細胞質(zhì)膜、內(nèi)質(zhì)網(wǎng)膜、胚乳貯藏囊泡、細胞骨架、線粒體、染色質(zhì)、細胞核、細胞質(zhì)、過氧化物酶體、油體及液泡膜等部位，N-末端可變域是CDPK亞細胞定位和功能的關(guān)鍵[18]。利用CDPK與綠色熒光蛋白(GFP)融合表達的方法，發(fā)現(xiàn)擬南芥CDPK幾乎全部定位于質(zhì)膜，少數(shù)定位于核。大多擬南芥CDPK具有專一的膜結(jié)合位點，即N-末端可變域的?；稽c，其中豆蔻?；稽c與靶細胞膜形成一個不可逆轉(zhuǎn)的松散的結(jié)合，而棕櫚?；稽c則具有可逆的穩(wěn)定的膜錨定結(jié)合[19]。豆蔻?；稽c上的甘氨酸突變?yōu)楸彼?如AtCPK2、AtCPK3、AtCPK5、AtCPK6、AtCPK9和AtCPK13)使CDPK的膜結(jié)合能力降低[20]，棕櫚酰化位點突變則使亞細胞定位由膜向核轉(zhuǎn)變。另外，CDPK的亞細胞定位也受非生物脅迫的影響，如冰草(Agropyroncristatum)McCPK1響應(yīng)低溫脅迫而改變亞細胞定位，由定位于質(zhì)膜向定位于細胞核、內(nèi)質(zhì)網(wǎng)(ER)和肌動蛋白絲轉(zhuǎn)變[21]。

3 CDPK的靶蛋白

了解CDPK在植物體內(nèi)作用的靶蛋白，對研究其功能具有重要意義。到目前為止，已發(fā)現(xiàn)多種CDPK作用的底物(表1)，它們參與多種細胞過程，如初級和次級代謝、脅迫響應(yīng)、離子和水分運輸、轉(zhuǎn)錄過程、信號轉(zhuǎn)導(dǎo)途徑等。

4 CDPK與14-3-3蛋白的相互作用

在上述CDPK的靶蛋白中，14-3-3蛋白因其與CDPK之間的交叉磷酸化作用受到學者的廣泛關(guān)注。14-3-3蛋白既與磷酸化的CDPK結(jié)合也可被CDPK磷酸化，在真核生物中通過磷酸化及與蛋白質(zhì)互作調(diào)節(jié)多種生物學過程。

4.1 14-3-3蛋白對CDPK的調(diào)節(jié)

14-3-3蛋白對CDPK的活性有直接調(diào)節(jié)作用。擬南芥AtCPK1是第1個被發(fā)現(xiàn)的受14-3-3蛋白調(diào)控的CDPK[45]，但在體外實驗中，AtCPK1未能與14-3-3ω蛋白相結(jié)合，因此推測AtCPK1和14-3-3ω之間的相互作用可能發(fā)生在胞內(nèi)[46]，作用位點仍有待鑒定。14-3-3蛋白除了參與CDPK活性的直接調(diào)節(jié)外，還可以控制CDPK的穩(wěn)定性。擬南芥AtCPK3已被證實為14-3-3的相互作用蛋白[47-48]，盡管體外實驗顯示14-3-3蛋白并不影響AtCPK3的激酶活性，但近期研究發(fā)現(xiàn)，饑餓誘導(dǎo)脅迫的AtCPK3在與14-3-3蛋白質(zhì)相互作用喪失后被選擇性切割[49]，證明擬南芥細胞內(nèi)的14-3-3與AtCPK3的互作可以穩(wěn)定AtCPK3的活性。此外，在擬南芥細胞程序性死亡過程中，壞死性真菌產(chǎn)生的毒素FB1(Fumonisin B1)誘導(dǎo)擬南芥中程序性死亡因子PHS(phytosphingosine)表達，PHS水平的增加導(dǎo)致胞內(nèi)Ca2+升高，從而激活A(yù)tCPK3活性并磷酸化14-3-3蛋白質(zhì)，14-3-3的磷酸化使得14-3-3/AtCPK3復(fù)合體解離，AtCPK3被切割[50-52]，這一過程進一步證實了14-3-3蛋白對CDPK具有保護作用。

4.2 CDPK對14-3-3蛋白的調(diào)節(jié)

14-3-3蛋白不僅結(jié)合并調(diào)節(jié)磷酸化后的CDPK，它們自身也被磷酸化。動植物14-3-3s的各個位點都有可能被磷酸化，磷酸化在調(diào)節(jié)14-3-3與靶蛋白的相互作用過程中發(fā)揮了極其重要的作用[53-54]。在動物細胞中，程序性死亡因子PHS誘導(dǎo)兩種不同的蛋白激酶PKA(protein kinase A)和PKCδ(protein kinase Cδ)識別并磷酸化14-3-3ζ二聚體界面處的絲氨酸殘基(Ser58)，Ser58磷酸化破壞14-3-3s的二聚體結(jié)構(gòu)，從而完成了細胞凋亡過程(圖2)；在植物細胞中，與過氧化物酶體結(jié)合的AtCPK1和AtCPK3、AtCPK6、AtCPK8、AtCPK24、AtCPK28均可在多個位點磷酸化蛋白14-3-3χ和14-3-3ε[24]，其中對14-3-3χ和14-3-3ε蛋白磷酸化最快速的是AtCPK3和AtCPK28，因為二者N-末端可變域序列具有高度的同源性。

表1 已有文獻報道的CDPK在植物體內(nèi)作用的靶蛋白及其調(diào)控作用Table 1 The target protein and its regulatory role of CDPK

5 CDPK/14-3-3復(fù)合體的活性調(diào)控作用

擬南芥具有13個14-3-3蛋白編碼基因，在植物發(fā)育的不同階段和不同組織中差異表達。CDPK/14-3-3復(fù)合體在植物信號通路中協(xié)同作用，共同調(diào)控代謝、激素信號傳導(dǎo)和氣孔運動，以及對非生物和生物脅迫的響應(yīng)等重要的生理生化過程[55-56]。

5.1 CDPK/14-3-3復(fù)合體在初級代謝過程中的活性調(diào)控作用

當光合作用不活躍時，擬南芥硝酸還原酶NR(nitrate reductase)在絲氨酸殘基(Ser534)處被AtCPK17或AtCPK28磷酸化，14-3-3蛋白通過結(jié)合葉片中磷酸化的NR，阻止亞硝酸鹽的產(chǎn)生，使其不能進一步還原成銨，最終導(dǎo)致NR失活[24,42]。菠菜(Spinaciaoleracea)硝酸還原酶SoNR則在絲氨酸殘基(Ser543)處被相關(guān)蛋白激酶SnRK(SNF1-related protein kinase)磷酸化后與抑制性蛋白14-3-3結(jié)合，由于擬南芥AtCPK3與菠菜SnRK具有非常相似的氨基酸序列，因此擬南芥AtCPK3能夠使菠菜NR磷酸化[41]。14-3-3蛋白也可通過結(jié)合被AtCPK3磷酸化的擬南芥6-磷酸果糖-2-激酶F2KP(fructose-2,6-bisphosphatase)參與碳循環(huán)過程[25,46]。

圖2 PHS誘導(dǎo)的細胞程序性死亡過程[52]Fig.2 PHS-induced programmed cell death process[52] PKA: 蛋白激酶A Protein kinase A; PKC: 蛋白激酶C Protein kinase C; P: 磷酸化Phosphorylation.

5.2 CDPK/14-3-3復(fù)合體在植物激素合成過程中的活性調(diào)控作用

CDPK與赤霉素(GA)信號傳導(dǎo)存在緊密的聯(lián)系。1995年，在水稻中首次發(fā)現(xiàn)由GA誘導(dǎo)的未知的膜定位CDPK[43]。10年后，Abbasi等[56]研究顯示，OsCDPK13在轉(zhuǎn)錄水平響應(yīng)GA而被活化；擬南芥AtCPK28為GA和茉莉酸(JA)的調(diào)節(jié)劑，AtCPK28突變體改變了維持GA穩(wěn)態(tài)的關(guān)鍵酶的表達，表現(xiàn)出枝條和葉柄生長緩慢的特性[57-58]；煙草中參與GA生物合成的反饋調(diào)節(jié)因子芽生長抑制因子RSG(repression of shoot growth)被鑒定為NtCDPK1的直接靶標，RSG通過控制GA生物合成基因的轉(zhuǎn)錄來維持GA穩(wěn)態(tài)。RSG在細胞質(zhì)基質(zhì)和細胞核之間來回移動，當GA濃度降低時，RSG轉(zhuǎn)入細胞核中激活GA合成基因的轉(zhuǎn)錄，合成的GA誘導(dǎo)細胞質(zhì)基質(zhì)中游離Ca2+增加，從而導(dǎo)致NtCDPK1構(gòu)象發(fā)生改變，促進RSG在S114片段磷酸化并與14-3-3蛋白結(jié)合。NtCDPK1與RSG和14-3-3蛋白質(zhì)形成異源三聚體并作為支架和媒介促進磷酸化后的RSG與14-3-3形成二聚體，RSG/14-3-3復(fù)合體從NtCDPK1脫落后，細胞質(zhì)基質(zhì)中的RSG失活，核內(nèi)靶基因的活化被抑制[43,58-59](圖3)。

圖3 CDPK和14-3-3蛋白對GA穩(wěn)態(tài)的調(diào)控[52]Fig.3 The regulation of CDPK and 14-3-3 protein on GA homeostasis[52] N: 細胞核Nucleus; C: 細胞質(zhì)基質(zhì)Cytoplasmic matrix; P: 磷酸化Phosphorylation.下同The same below.

在植物乙烯的生物合成途徑中，限速酶1-氨基環(huán)丙烷-1-羧酸合酶ACS(1-aminocyclopropane-1-carboxylic acid ACC synthase)家族起到了催化調(diào)控的作用。ACS因其C-末端存在不同的序列被分為3種類型，14-3-3s與所有類型的ACS均可以相互作用[39]。擬南芥AtACS7是一種3型ACS，可以被AtCPK16在3個位點(Ser216、Thr296和Ser299)處磷酸化，然后在細胞質(zhì)基質(zhì)中與14-3-3ω直接相互作用形成蛋白復(fù)合體[60-61]，AtCPK16和14-3-3ω的協(xié)同作用增加了AtACS7的穩(wěn)定性。

5.3 CDPK/14-3-3復(fù)合體在開花過程中的活性調(diào)控作用

凝膠內(nèi)激酶測定結(jié)果顯示，磷脂酰乙醇胺結(jié)合蛋白開花位點D(AtFD)T282片段可能是AtCPK6和AtCPK33的底物[62]，磷酸化后的AtFD與14-3-3蛋白形成復(fù)合體，與開花位點T(AtFT)相互作用激活花分生組織基因如擬南芥花分生組織決定基因AP1(Apetala 1)的轉(zhuǎn)錄，從而促進開花過程[63]。而AtCPK33突變體則表現(xiàn)出輕度延遲開花的現(xiàn)象(圖4)。

6 CDPK的生物學功能

6.1 CDPKs在植物發(fā)育中的作用

圖4 CDPK調(diào)控開花過程[4]Fig.4 CDPK control flowering process[4]

圖5 CDPK對鈣信號傳導(dǎo)的調(diào)控[4]Fig.5 Regulation of calcium signaling of CDPK[4]

CDPK是植物所有主要發(fā)育過程包括傳粉、生長、開花和衰老的調(diào)控者。許多擬南芥CDPK(AtCPK2、AtCPK14、AtCPK16、AtCPK17、AtCPK20、AtCPK24、AtCPK26和AtCPK34)主要在花粉中表達，表明它們參與花粉發(fā)育或花粉管伸長。最近的研究表明，擬南芥中的兩種CDPK，依賴Ca2+的AtCPK11和不依賴于Ca2+的AtCPK24可以形成級聯(lián)信號，AtCPK11磷酸化AtCPK24 N-末端結(jié)構(gòu)域，在花粉管特異性鉀離子內(nèi)流通道SPIK(shaker pollen inward K+)定位的質(zhì)膜處相互作用并抑制其活性，從而對花粉管伸長進行負調(diào)節(jié)作用。此外，其他兩種花粉特異性CDPK，AtCPK17和AtCPK34也定位于細胞質(zhì)膜，推測可能與花粉伸長過程相關(guān)，研究發(fā)現(xiàn)AtCPK17/AtCPK34雙突變體在花粉管的頂端極化生長表達上存在缺陷，而單個的突變則正常表達[64-65]。

6.2 CDPK對鈣信號傳導(dǎo)的調(diào)控

在產(chǎn)生鈣信號過程中，擬南芥內(nèi)質(zhì)網(wǎng)膜和細胞質(zhì)膜上分別具有自我抑制型Ca2+泵ACA2(autoinhibted calcium ATPase 2)和ACA8(autoinhibted calcium ATPase 8)，這兩種Ca2+泵調(diào)控Ca2+泵入內(nèi)質(zhì)網(wǎng)腔或泵出細胞，維持細胞質(zhì)基質(zhì)中較低的Ca2+濃度，二者均可以直接被CDPK磷酸化。Giacometti等[66]研究表明，AtCPK16定位于質(zhì)膜，因此在質(zhì)膜內(nèi)磷酸化AtACA8。AtCPK1可以與過氧化物酶體和微粒體結(jié)合，推測與過氧化物酶體相結(jié)合的AtCPK1/P復(fù)合體可作用于AtACA2和AtACA8；而ER上可衍生出微粒體，因此推測與微粒體相結(jié)合的AtCPK1/LB復(fù)合體可能與AtACA2連接并調(diào)控其作用(圖5)。

6.3 CDPK參與植物非生物脅迫應(yīng)答

研究表明，CDPK參與植物非生物脅迫反應(yīng)，低溫[67]、光[68]、干旱[69]、鹽害[70]、低滲透[71]、營養(yǎng)饑餓等多種環(huán)境因子以及赤霉素GA、生長素、細胞分裂素等激素的誘導(dǎo)都能引起CDPK基因的特異性表達，如表2所列。

表2 不同植物中響應(yīng)非生物脅迫的CDPKTable 2 CDPK responsed to abiotic stresses in different plants

6.4 CDPK與氣孔運動

ABA依賴性的氣孔閉合是防止干旱脅迫期間水分流失的關(guān)鍵機制[80]，許多CDPK參與到氣孔運動的調(diào)控過程中[81]。在擬南芥中，離子通道AtKAT1和AtKAT2以及其他K+轉(zhuǎn)運蛋白具有調(diào)節(jié)氣孔開放的功能[54,80]。AtCPK1則激活液泡膜上的Cl-通道，導(dǎo)致Cl-內(nèi)流；AtCPK1介導(dǎo)的Cl-內(nèi)流、AtKAT1/AtKAT2介導(dǎo)的K+內(nèi)流、陰離子通道SLAC1(slowtype anion-associated 1)與SLAH3(SLAC1 homologue 3)介導(dǎo)的陰離子(Cl-、NO3-)外流三者協(xié)同作用，提高了保衛(wèi)細胞的滲透勢，導(dǎo)致保衛(wèi)細胞膨脹，氣孔打開(圖6左)；AtCPK3、AtCPK13是保衛(wèi)細胞中主要表達的Ca2+敏感性CDPK，磷酸化AtKAT1和AtKAT2[82]，從而抑制K+內(nèi)流過程。Sato等[83]的研究表明，AtSnRK2和擬南芥AtCPK4、AtCPK13可以在細胞內(nèi)磷酸化AtKAT1的C-末端片段T306和T308，表明擬南芥CDPK和SnRK2之間可能存在一致的靶位點；T306的突變導(dǎo)致AtKAT1和AtKAT2通道活性受損。ABA信號傳導(dǎo)產(chǎn)生激活的AtCPK3、AtCPK6、AtCPK21和AtCPK23以及AtOST1的活性氧(ROS)和Ca2+信號，這些激酶在保衛(wèi)細胞離子通道的調(diào)節(jié)中起主要作用，它們激活A(yù)tSLAC1和AtSLAH3陰離子通道，促進陰離子的流出[84-86]。此外，AtCPK3、AtCPK4、AtCPK5、AtCPK11和AtCPK29在液泡外磷酸化雙孔鉀離子通道TPK1(tandem-pore K+channel 1)，磷酸化的AtTPK1與14-3-3蛋白質(zhì)形成蛋白復(fù)合體介導(dǎo)液泡中的K+流出[87]。質(zhì)膜質(zhì)子泵的抑制依賴于Ca2+的陰離子通道的活化導(dǎo)致質(zhì)膜去極化，激活保衛(wèi)細胞外向整流型K+通道GORK(guard cell outward-rectifying K+channel)，K+外流。最終，保衛(wèi)細胞內(nèi)溶質(zhì)的流出導(dǎo)致其膨脹程度降低，氣孔閉合(圖6右)。

圖6 CDPK對氣孔運動過程的調(diào)控[4]Fig.6 Regulation of stomatal movement of CDPK[4]

7 結(jié)語

到目前為止，CDPK的結(jié)構(gòu)、亞細胞定位以及調(diào)控機制等研究已經(jīng)比較成熟，越來越多CDPK家族成員的新功能已經(jīng)被發(fā)現(xiàn)，但其作用底物和抑制劑還有待于進一步的研究，如弓形蟲寄生病的治療。弓形蟲是一種常見的原生動物，幾乎可以感染所有溫血動物和人類，目前尚缺乏有效的治療藥物，但由于弓形蟲中有CDPK，而人體并無CDPK，因此CDPK專一性抑制劑可能成為弓形蟲治療的新型藥物。此外，CDPK和14-3-3蛋白質(zhì)之間存在復(fù)雜的調(diào)控網(wǎng)絡(luò)。如本研究所述，CDPK不僅磷酸化14-3-3蛋白,其本身也被14-3-3磷酸化，盡管近年的多項研究都顯示出CDPK/14-3-3復(fù)合體在植物活性調(diào)控過程中的重要作用，但CPDKs與14-3-3s通過磷酸化的交叉調(diào)節(jié)過程仍不清晰，有待于國內(nèi)外學者的進一步探索。

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