Angiotensin II–Inducible Platelet-Derived Growth Factor-D Transcription Requires Specific Ser/Thr Residues in the Second Zinc Finger Region of Sp1

Abstract

Abstract-Sp1, the first identified and cloned transcription factor, regulates gene expression via multiple mechanisms including direct protein-DNA interactions, protein-protein interactions, chromatin remodeling, and maintenance of methylation-free CpG islands. Sp1 is itself regulated at different levels, for example, by glycosylation, acetylation, and phosphorylation by kinases such as the atypical protein kinase C-. Although Sp1 controls the basal and inducible regulation of many genes, the posttranslational processes regulating its function and their relevance to pathology are not well understood. Here we have used a variety of approaches to identify 3 amino acids (Thr668, Ser670, and Thr681) in the zinc finger domain of Sp1 that are modified by PKC-and have generated novel anti-peptide antibodies recognizing the PKC--phosphorylated form of Sp1. Angiotensin II, which activates PKC-phosphorylation (at Thr410) via the angiotensin II type 1 receptor, stimulates Sp1 phosphorylation and increases Sp1 binding to the platelet-derived growth factor-D promoter. All 3 residues in Sp1 (Thr668, Ser670, and Thr681) are required for Sp1-dependent platelet-derived growth factor-D activation in response to angiotensin II. Immunohistochemical analysis revealed that phosphorylated Sp1 is expressed in smooth muscle cells of human atherosclerotic plaques and is dynamically expressed together with platelet-derived growth factor-D in smooth muscle cells of the injured rat carotid artery wall. This study provides new insights into the regulatory mechanisms controlling the PKC--phospho-Sp1 axis and angiotensin II-inducible gene expression. (Circ Res. 2008;102:e38-e51.)

Sp1 Phosphorylation and Its Regulation of Gene Transcription

“…Mutation of Thr579 in the consensus CKII site did not eliminate CKII phosphorylation of Sp1, though it did perturb CKII's ability to inhibit Sp1 binding to DNA in vitro. Treatment of K562 cells with okadaic acid to inhibit endoge- We recently demonstrated that Thr668, along with Ser670 (and Thr681), is a target of phosphorylation by PKC-using a combination of approaches including in vitro peptide and protein phosphorylation analysis with Ala mutant counterparts, mass spectrometry phosphopeptide analysis, and coimmunoprecipitation analysis (63). This follows investigations by Pal et al demonstrating that PKC-binds to and phosphorylates the zinc finger region of Sp1 (49).…”

“…This is the first demonstration of phosphorylated Sp1 in diseased animal and human tissue (63). We previously showed that activated PKC-(pThr410) is also expressed in atherosclerotic plaques in the context of the Fas ligand (29).…”

“…Sp1 is phosphorylated by various kinases at different sites within the protein (Table 2), and the influence of these modifications on the function of the transcription factor is only beginning to be understood. Kinases regulating Sp1 binding or transactivity include cyclin-dependent kinase (CDK) (17), atypical protein kinase C-(PKC-) (30,52,63), extracellular signal-regulated kinase (ERK) (3,46), casein kinase II (CKII) (1), and DNA-dependent protein kinase (11). Human Sp1 (NCBI accession number P08047) has 61 putative phosphorylation sites, with 48 of these residues being Ser, 10 Thr, and 3 Tyr (from NetPhos 2.0; data not shown).…”

“…For example, although we understand that certain kinases and residues in Sp1 mediate Sp1-dependent transcription, as in the case of (49), it is unclear whether or how Sp1 phosphorylation influences chromatin remodeling, conformational changes, the recruitment of coactivators or repressors, and other posttranslational modifications. The availability of phospho-specific antibodies, such as those targeting pSer59 (65), pThr453 (46), p668/670 (63), p681 (63), and pThr739 (46), will serve as important tools in future investigations to more precisely define the roles of phosphorylation in the functions of this important transcription factor in health and disease.…”

Association of platelet-derived growth factor-D gene polymorphism with ischemic stroke in a Chinese case–control study

“…Previous study had shown that angiotensin II by the angiotensin II type 1 receptor stimulates Sp1 phosphorylation and increases Sp1 binding to the PDGF-D [17,18]. PDGF-D by binding with the b-receptor but not areceptor [19,20] contributes to the pathophysiological process in the development and progression of cell proliferation and accumulation of extracellular matrix.…”