Constructing a plasmid encoding the human PKD1 protein (25-636aa) and the N-terminal 6xHis-tag and C-terminal 6xHis-tag initiates the general approach for generating the recombinant human PKD1 protein. Transfection of the plasmid into E.coli cells creates the E.coli cells containing plasmid, which are cultured and induced for protein expression. Subsequently, the protein is purified through affinity purification, and SDS-PAGE analysis is undertaken to confirm the presence and assess the purity of the protein. The protein's purity exceeds 85%.
Protein kinase D1 (PKD1) serves as a critical player in cellular signaling, impacting various cellular functions. It oversees multiple signaling routes like NF-κB, ERK1/2, and Akt while damping down JNK and p38, thus governing cell survival and growth [1]. PKD1's actions hinge on its interactions with other proteins, which help it position itself strategically near upstream kinases, lipid regulators, and specific substrates, influencing its subcellular localization and activation [2].
In cancer, PKD1's involvement in promoting epidermal growth factor signaling has been noted, potentially fueling cell proliferation, though its precise role in cancer remains a topic of debate [3]. Being the founding member of a family of Ser/Thr kinases, PKD1 oversees various cellular processes like growth, apoptosis, adhesion, motility, and angiogenesis [4]. Studies have highlighted PKD1's pro-proliferative and anti-differentiation effects in mouse keratinocytes [5]. Moreover, PKD1 phosphorylation has been found to safeguard dopaminergic neurons during oxidative stress, suggesting a role in neuroprotection [6].
PKD1's implications stretch to numerous cancers like gastric, pancreatic, and prostate cancer, where it influences cell movement, invasion, and metabolic adaptation. In pancreatic cancer, it's a target of the KRas-NF-κB pathway [7][8][9][10]. In endothelial cells, PKD1 emerges as a pivotal regulator of angiogenesis-related signaling pathways [11]. It's also linked with cardiac hypertrophy, facilitating nuclear import during this condition [12]. Additionally, PKD1 restrains cell proliferation in prostate cancer by affecting the secretion of matrix metalloproteinases [13]. Cholecystokinin signaling in pancreatic acini activates PKD1 through both PKC-δ and PKC-independent pathways, indicating its involvement in protein trafficking, cell growth, and programmed cell death [14].
References:
[1] L. Zhang, Z. Zhao, S. Xu, M. Tandon, C. LaValle, F. Denget al., Androgen suppresses protein kinase d1 expression through fibroblast growth factor receptor substrate 2 in prostate cancer cells, Oncotarget, vol. 8, no. 8, p. 12800-12811, 2017. https://doi.org/10.18632/oncotarget.14536
[2] Z. Li, C. Zhang, L. Chen, G. Li, Q. Liu, K. Balajiet al., E‐cadherin facilitates protein kinase d1 activation and subcellular localization, Journal of Cellular Physiology, vol. 231, no. 12, p. 2741-2748, 2016. https://doi.org/10.1002/jcp.25382
[3] C. Legay, S. Doublier, S. Babajko, & J. Ricort, Protein kinase d1 overexpression potentiates epidermal growth factor signaling pathway in mcf-7 cells, Molecular Biology Reports, vol. 50, no. 4, p. 3641-3651, 2023. https://doi.org/10.1007/s11033-023-08300-z
[4] W. Qiu, F. Zhang, & S. Steinberg, The protein kinase d1 cooh terminus: marker or regulator of enzyme activity?, Ajp Cell Physiology, vol. 307, no. 7, p. C606-C610, 2014. https://doi.org/10.1152/ajpcell.00155.2014
[5] I. Youssef and J. Ricort, Deciphering the role of protein kinase d1 (pkd1) in cellular proliferation, Molecular Cancer Research, vol. 17, no. 10, p. 1961-1974, 2019. https://doi.org/10.1158/1541-7786.mcr-19-0125
[6] A. Asaithambi, M. Ay, H. Jin, A. Gosh, V. Anantharam, & A. Kanthasamy, Protein kinase d1 (pkd1) phosphorylation promotes dopaminergic neuronal survival during 6-ohda-induced oxidative stress, Plos One, vol. 9, no. 5, p. e96947, 2014. https://doi.org/10.1371/journal.pone.0096947
[7] M. Kim, H. Jang, J. Kim, S. Noh, K. Song, J. Choet al., Epigenetic inactivation of protein kinase d1 in gastric cancer and its role in gastric cancer cell migration and invasion, Carcinogenesis, vol. 29, no. 3, p. 629-637, 2007. https://doi.org/10.1093/carcin/bgm291
[8] S. Kumari, S. Khan, R. Sekhri, H. Mandil, S. Behrman, M. Yallapuet al., Protein kinase d1 regulates metabolic switch in pancreatic cancer via modulation of mtorc1, British Journal of Cancer, vol. 122, no. 1, p. 121-131, 2019. https://doi.org/10.1038/s41416-019-0629-9
[9] H. Döppler, R. Panayiotou, E. Reid, W. Maimo, L. Bastea, & P. Storz, The prkd1 promoter is a target of the kras-nf-κb pathway in pancreatic cancer, Scientific Reports, vol. 6, no. 1, 2016. https://doi.org/10.1038/srep33758
[10] V. Sundram, S. Chauhan, & M. Jaggi, Emerging roles of protein kinase d1 in cancer, Molecular Cancer Research, vol. 9, no. 8, p. 985-996, 2011. https://doi.org/10.1158/1541-7786.mcr-10-0365
[11] C. Ha and Z. Jin, Protein kinase d1, a new molecular player in vegf signaling and angiogenesis, Molecules and Cells, vol. 28, no. 1, p. 1-6, 2009. https://doi.org/10.1007/s10059-009-0109-9
[12] Y. Sin, T. Martin, L. Wills, S. Currie, & G. Baillie, Small heat shock protein 20 (hsp20) facilitates nuclear import of protein kinase d 1 (pkd1) during cardiac hypertrophy, Cell Communication and Signaling, vol. 13, no. 1, 2015. https://doi.org/10.1186/s12964-015-0094-x
[13] M. Biswas, C. Du, C. Zhang, J. Straubhaar, L. Languino, & K. Balaji, Protein kinase d1 inhibits cell proliferation through matrix metalloproteinase-2 and matrix metalloproteinase-9 secretion in prostate cancer, Cancer Research, vol. 70, no. 5, p. 2095-2104, 2010. https://doi.org/10.1158/0008-5472.can-09-4155
[14] M. Berna, K. Hoffmann, J. Tapia, M. Thill, A. Pace, S. Manteyet al., Cck causes pkd1 activation in pancreatic acini by signaling through pkc-δ and pkc-independent pathways, Biochimica Et Biophysica Acta (Bba) - Molecular Cell Research, vol. 1773, no. 4, p. 483-501, 2007. https://doi.org/10.1016/j.bbamcr.2006.12.008
