Mitogen-activated protein kinases(MAPKs)are a family of proteins that constitute signaling pathways involved in processes that control gene expression,cell division, cell survival,apoptosis,metabolism,differentiation ...Mitogen-activated protein kinases(MAPKs)are a family of proteins that constitute signaling pathways involved in processes that control gene expression,cell division, cell survival,apoptosis,metabolism,differentiation and motility.The MAPK pathways can be divided into conventional and atypical MAPK pathways.The first group converts a signal into a cellular response through a relay of three consecutive phosphorylation events exerted by MAPK kinase kinases,MAPK kinase,and MAPK.Atypical MAPK pathways are not organized into this three-tiered cascade.MAPK that belongs to both conventional and atypical MAPK pathways can phosphorylate both non-protein kinase substrates and other protein kinases.The latter are referred to as MAPK-activated protein kinases.This review focuses on one such MAPK-activated protein kinase,MAPK-activated protein kinase 5(MK5)or p38-regulated/activated protein kinase(PRAK).This protein is highly conserved throughout the animal kingdom and seems to be the target of both conventional and atypical MAPK pathways.Recent findings on the regulation of the activity and subcellular localization,bona fide interaction partners and physiological roles of MK5/PRAK are discussed.展开更多
Chemosensory proteins (CSPs) are ubiquitous soluble small proteins isolated from sensory organs thought to be involved in chemical communication. Here we report the first cDNA of CSPs, called Ac-ASP3, cloned and chara...Chemosensory proteins (CSPs) are ubiquitous soluble small proteins isolated from sensory organs thought to be involved in chemical communication. Here we report the first cDNA of CSPs, called Ac-ASP3, cloned and characterized from antennas of adult worker bees in Chinese honeybee, Apis cerana cerana. The Ac-ASP3 cDNA comprises 2 exons, with an ORF of 393-bp encoding 130 aa. Protein signature analyses show that the protein consists of four conserved cysteines and a signal peptide with 19 aa in the N-terminal sequence. The deduced protein sequence shares high homology with Am-ASP3 of Apis mellifera and low similarity with other species of insects. Immunocytochemical localization shows that Ac-ASP3 is only specifically expressed on the antenna contact chemosensilla such as sensilla trichodea B and sensilla basiconica, whereas Ac-ASP3 is scarcely expressed on olfactory chemosensilla such as sensilla placodea. Real-time PCR of Ac-ASP3 transcripts shows that Ac-ASP3 is highly expressed on wings and legs, but expression is lower on antenna. Temporal expression patterns suggest that Ac-ASP3 is expressed during the period of pupa and adults from 1-d to 6-d stages when bees act as house bees, cleaning the comb and taking care of the queen and larvae in comb. The above evidence suggests that Ac-ASP3 is unique in species and is generally not involved in olfaction during searching for honey and pollen. Rather, the protein seems to function in recognition of chemosensory substances on bees' cuticle and mechanical movement of antenna.展开更多
By screening tobacco cDNA library with MCK1 as a probe, we isolated a cDNA clone NtCPK5 （accession number AY971376）, which encodes a typical calcium-dependent protein kinase. Sequence analyses indicated that NtCPK5 ...By screening tobacco cDNA library with MCK1 as a probe, we isolated a cDNA clone NtCPK5 （accession number AY971376）, which encodes a typical calcium-dependent protein kinase. Sequence analyses indicated that NtCPK5 is related to both CPKs and CRKs superfamilies and has all of the three conserved domains of CPKs. The biochemical activity of NtCPK5 was calcium-dependent. NtCPK5 had Vmax and Km of 526nmol/min/mg and 210μg/ml respectively with calf thymus histone （fraction Ⅲ, abbreviated to histone Ⅲs） as substrate. For substrate syntide-2, NtCPK5 showed a higher Vmax of 2008 nmol/min/mg and a lower Km of 30μM. The K0.5 of calcium activation was 0.04μM or 0.06μM for histone Ⅲs or syntide-2 respectively. The putative myristoylation and palmitoylation consensus sequence of NtCPK5 suggests that it could be a membrane-anchoring protein. Indeed, our transient expression experiments with wild type and mutant forms of NtCPK5/GFP fusion proteins showed that NtCPK5 was localized to the plasma membrane of onion epidermal cells and that the localization required the N-terminal acylation sites of NtCPK5/GFP. Taking together, our data have demonstrated the biochemical characteristics of a novel protein NtCPK5 and its subcellular localization as a membrane-anchoring protein.展开更多
Targeting of the synthesized polypeptide in the cells is an important research field in modern cell biology. Cowpea trypsin inhibitor (cpti) gene has been modified and a fusion protein gene (sck) was produced by fusin...Targeting of the synthesized polypeptide in the cells is an important research field in modern cell biology. Cowpea trypsin inhibitor (cpti) gene has been modified and a fusion protein gene (sck) was produced by fusing a signal peptide sequence at cpti 5' end and an endoplasm reticulum (ER) retention signal peptide at cpti3' end respectively. The signal peptide can direct the newly synthesized polypeptide into ER, while ER retention signal can make the protein retained in the ER and its derivative protein body. ELISA test indicated that the accumulation level of foreign CpTI protein in sck transgenic tobacco (Nicotiana tabacum L.) was two times higher than cpti transgenic tobaccos and some individuals were four times higher. At the same time, sck transgenic tobacco has a high resistance to Lepidoptera pest due to the increased accumulation level of foreign CpTI protein. The strategy of foreign protein targeting can be used to increase the accumulation level of foreign protein in transgenic plants and can be widely applied to other related research field in plant genetic engineering.展开更多
文摘Mitogen-activated protein kinases(MAPKs)are a family of proteins that constitute signaling pathways involved in processes that control gene expression,cell division, cell survival,apoptosis,metabolism,differentiation and motility.The MAPK pathways can be divided into conventional and atypical MAPK pathways.The first group converts a signal into a cellular response through a relay of three consecutive phosphorylation events exerted by MAPK kinase kinases,MAPK kinase,and MAPK.Atypical MAPK pathways are not organized into this three-tiered cascade.MAPK that belongs to both conventional and atypical MAPK pathways can phosphorylate both non-protein kinase substrates and other protein kinases.The latter are referred to as MAPK-activated protein kinases.This review focuses on one such MAPK-activated protein kinase,MAPK-activated protein kinase 5(MK5)or p38-regulated/activated protein kinase(PRAK).This protein is highly conserved throughout the animal kingdom and seems to be the target of both conventional and atypical MAPK pathways.Recent findings on the regulation of the activity and subcellular localization,bona fide interaction partners and physiological roles of MK5/PRAK are discussed.
基金Supported by the National Natural Science Foundation of China (Grant No. 30270896)
文摘Chemosensory proteins (CSPs) are ubiquitous soluble small proteins isolated from sensory organs thought to be involved in chemical communication. Here we report the first cDNA of CSPs, called Ac-ASP3, cloned and characterized from antennas of adult worker bees in Chinese honeybee, Apis cerana cerana. The Ac-ASP3 cDNA comprises 2 exons, with an ORF of 393-bp encoding 130 aa. Protein signature analyses show that the protein consists of four conserved cysteines and a signal peptide with 19 aa in the N-terminal sequence. The deduced protein sequence shares high homology with Am-ASP3 of Apis mellifera and low similarity with other species of insects. Immunocytochemical localization shows that Ac-ASP3 is only specifically expressed on the antenna contact chemosensilla such as sensilla trichodea B and sensilla basiconica, whereas Ac-ASP3 is scarcely expressed on olfactory chemosensilla such as sensilla placodea. Real-time PCR of Ac-ASP3 transcripts shows that Ac-ASP3 is highly expressed on wings and legs, but expression is lower on antenna. Temporal expression patterns suggest that Ac-ASP3 is expressed during the period of pupa and adults from 1-d to 6-d stages when bees act as house bees, cleaning the comb and taking care of the queen and larvae in comb. The above evidence suggests that Ac-ASP3 is unique in species and is generally not involved in olfaction during searching for honey and pollen. Rather, the protein seems to function in recognition of chemosensory substances on bees' cuticle and mechanical movement of antenna.
基金supported in part by the National Natural Science Foundation of China(Grant No.30230050)the Program for Changjiang Scholars and Innovative Research Team in University to Ying Tang LU.
文摘By screening tobacco cDNA library with MCK1 as a probe, we isolated a cDNA clone NtCPK5 （accession number AY971376）, which encodes a typical calcium-dependent protein kinase. Sequence analyses indicated that NtCPK5 is related to both CPKs and CRKs superfamilies and has all of the three conserved domains of CPKs. The biochemical activity of NtCPK5 was calcium-dependent. NtCPK5 had Vmax and Km of 526nmol/min/mg and 210μg/ml respectively with calf thymus histone （fraction Ⅲ, abbreviated to histone Ⅲs） as substrate. For substrate syntide-2, NtCPK5 showed a higher Vmax of 2008 nmol/min/mg and a lower Km of 30μM. The K0.5 of calcium activation was 0.04μM or 0.06μM for histone Ⅲs or syntide-2 respectively. The putative myristoylation and palmitoylation consensus sequence of NtCPK5 suggests that it could be a membrane-anchoring protein. Indeed, our transient expression experiments with wild type and mutant forms of NtCPK5/GFP fusion proteins showed that NtCPK5 was localized to the plasma membrane of onion epidermal cells and that the localization required the N-terminal acylation sites of NtCPK5/GFP. Taking together, our data have demonstrated the biochemical characteristics of a novel protein NtCPK5 and its subcellular localization as a membrane-anchoring protein.
文摘Targeting of the synthesized polypeptide in the cells is an important research field in modern cell biology. Cowpea trypsin inhibitor (cpti) gene has been modified and a fusion protein gene (sck) was produced by fusing a signal peptide sequence at cpti 5' end and an endoplasm reticulum (ER) retention signal peptide at cpti3' end respectively. The signal peptide can direct the newly synthesized polypeptide into ER, while ER retention signal can make the protein retained in the ER and its derivative protein body. ELISA test indicated that the accumulation level of foreign CpTI protein in sck transgenic tobacco (Nicotiana tabacum L.) was two times higher than cpti transgenic tobaccos and some individuals were four times higher. At the same time, sck transgenic tobacco has a high resistance to Lepidoptera pest due to the increased accumulation level of foreign CpTI protein. The strategy of foreign protein targeting can be used to increase the accumulation level of foreign protein in transgenic plants and can be widely applied to other related research field in plant genetic engineering.
