A phosphorylation state-specific antibody recognizes Hsp27, a novel substrate of protein kinase D.
ABSTRACT The use of phosphorylation state-specific antibodies has revolutionized the field of cellular signaling by Ser/Thr protein kinases. A more recent application of this technology is the development of phospho-specific antibodies that specifically recognize the consensus substrate phosphorylated motif of a given protein kinase. Here, we describe the development and use of such an antibody which is directed against the optimal phosphorylation motif of protein kinase D (PKD). A degenerate phosphopeptide library with fixed residues corresponding to the consensus LXR(Q/K/E/M)(M/L/K/E/Q/A)S*XXXX was used as an antigen to generate an antibody that recognizes this motif. We characterized the antibody by enzyme-linked immunosorbent assay and with immobilized peptide arrays and also detected immunoreactive phosphoproteins in HeLa cells stimulated with agonists known to activate PKD. Silencing PKD expression using RNA interference validated the specificity of this antibody immunoreactive against putative substrates. The antibody also detected the PKD substrates RIN1 and HDAC5. Knowledge of the PKD consensus motif also enabled us to identify Ser(82) in the human heat shock protein Hsp27 as a novel substrate for PKD. We term this antibody anti-PKD pMOTIF and predict that it will enable the discovery of novel PKD substrate proteins in cells.
Article: Phosphorylation of Ser78 of Hsp27 correlated with HER-2/neu status and lymph node positivity in breast cancer.[show abstract] [hide abstract]
ABSTRACT: Abnormal amplification/expression of HER-2/neu oncogene has been causally linked with tumorigenesis and metastasis in breast cancer and associated with shortened overall survival of patients. Recently, heat shock protein 27 (Hsp27) was reported to be highly expressed in HER-2/neu positive tumors and cell lines. However, putative functional links between phosphorylation of Hsp27 with HER-2/neu status and other clinicopathological features remain to be elucidated. Comparative phosphoproteomic studies of HER-2/neu positive and -negative breast tumors revealed that Hsp27, one of the identified phosphoproteins, was highly phosphorylated in HER-2/neu positive tumors. The extent of Hsp27 phosphorylation at its Ser15, Ser78 and Ser82 residues were further evaluated with site-specific antibodies in tumor samples by tissue lysate array- and tissue microarray-based analyses, and in the BT474 breast cancer cell line treated with heregulin alpha1 (HRG alpha1) or the p38 MAPK inhibitor, SB203580. The tissue lysate array study indicated that only the level of pSer78 in HER-2/neu positive tumors was more than 2-fold that in HER-2/neu negative tumors. Treatment of BT474 cells with HRG alpha1 and SB203580 indicated that Ser78 phosphorylation was mainly regulated by the HER-2/neu-p38 MAPK pathway. Immunohistochemical staining of sections from a tissue microarray with 97 breast tumors showed that positive staining of pSer78 significantly correlated with HER-2/neu (p = 0.004) and lymph node positivity (p = 0.026). This investigation demonstrated the significant correlation of enhanced phosphorylation of the Ser78 residue of Hsp27 with HER-2/neu and lymph node positivity in breast cancer.Molecular Cancer 02/2007; 6:52. · 3.99 Impact Factor
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ABSTRACT: Autophagy, a process in which cellular components are engulfed and degraded within double-membrane vesicles termed autophagosomes, has an important role in the response to oxidative damage. Here we identify a novel cascade of phosphorylation events, involving a network of protein and lipid kinases, as crucial components of the signaling pathways that regulate the induction of autophagy under oxidative stress. Our findings show that both the tumor-suppressor death-associated protein kinase (DAPk) and protein kinase D (PKD), which we previously showed to be phosphorylated and consequently activated by DAPk, mediate the induction of autophagy in response to oxidative damage. Furthermore, we map the position of PKD within the autophagic network to Vps34, a lipid kinase whose function is indispensable for autophagy, and demonstrate that PKD is found in the same molecular complex with Vps34. PKD phosphorylates Vps34, leading to activation of Vps34, phosphatydilinositol-3-phosphate (PI(3)P) formation, and autophagosome formation. Consistent with its identification as a novel inducer of the autophagic machinery, we show that PKD is recruited to LC3-positive autophagosomes, where it localizes specifically to the autophagosomal membranes. Taken together, our results describe PKD as a novel Vps34 kinase that functions as an effecter of autophagy under oxidative stress.Cell death and differentiation 11/2011; 19(5):788-97. · 8.24 Impact Factor
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ABSTRACT: The degree of phosphorylation of Myosin Light Chain 20 (MLC20) is a major determinant of force generation in smooth muscle. Myosin phosphatases (MP) all contain Protein Phosphatase 1 as catalytic subunits and are the major enzymes that dephosphorylate MLC20. MYPT1, the main regulatory subunit of MP in all smooth muscles, is a key convergence point of both contractile and relaxatory pathways. Combinations of regulatory mechanisms including isoform splicing, multiple phosphorylation sites and scaffolding proteins modulate MYPT1 activity with tissue- and agonist- specificities to affect contraction and relaxation. Other PP1 phosphatases that do not target myosin, as well as PP2A and PP2B dephosphorylate a range of proteins that affect smooth muscle contraction. This review discusses the role of phosphatases in smooth muscle contractility with a focus on MYPT1 in uterine smooth muscle. The myometrium shares characteristics of vascular and other visceral smooth muscles, yet during normal pregnancy undergoes hypertrophy, hyperplasia, quiescence and labor as normal physiology processes. The myometrium presents an accessible model for the study of normal and pathological smooth muscle function and a better understanding of myometrial physiology may allow the development of novel therapeutics for the many disorders of myometrial physiology from preterm labor to dysmenorrhea.AJP Cell Physiology 01/2013; · 3.54 Impact Factor