Growth factor modulated calmodulin-binding protein stimulates nuclear DNA synthesis in hemopoietic progenitor cells

Gp, Reddy; Reed, WC; Dh, Deacon; Quesenberry, PJ

doi:10.1021/bi00187a030

Cited by 14 publications

(7 citation statements)

References 41 publications

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“…6A, lane 4), suggesting that although CaM is essential, it alone is not sufficient, and other necessary nuclear proteins are removed by co-precipitation with antibody. In this context, calmodulin binding protein CaM-BP68, which is involved in CaM action (31,32), together with CaM and ER also failed to generate any ER⅐ERE complex in the reconstituted system (data not shown).…”

Section: Cam Depletion From Nuclear Extract Of Mda-mb-231 Cells Prevementioning

confidence: 92%

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Calmodulin Is Essential for Estrogen Receptor Interaction with Its Motif and Activation of Responsive Promoter

Biswas¹,

Reddy²,

Pickard³

et al. 1998

Journal of Biological Chemistry

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Calmodulin (CaM) has been reported to have affinity for the estrogen receptor (ER). Observations reported here reveal a direct physical interaction between purified CaM and ER. This direct ER-CaM interaction may be an initial event preceding the assembly of ER plus auxiliary proteins into the active ER complex with its DNA motif, the estrogen response element. We demonstrate that CaM is an integral component of this complex by using a system reconstituted from purified ER and nuclear extract from ER-negative breast cancer cells and also with ER-depleted nuclear extract of an ER-positive breast cancer cell line. Although CaM is essential for formation of this complex, it is not sufficient, suggesting roles also of auxiliary proteins. CaM also is functionally required for activation of an ER-responsive promoter, in the 17␤-estradiol-ER pathway of hormone action and regulation of 17␤-estradiol-responsive gene expression that is associated with proliferation of mammary epithelial cells. CaM1 plays a pivotal role in the proliferation of a variety of cells. Several observations indicate CaM involvement in estrogen regulation of breast cancer cell growth. Calcium homeostasis is lost (1), and expression of calcium binding proteins is modulated (2-4). There is an overall increase of Ca 2ϩ levels in human mammary tumors (5, 6), and CaM concentrations are 2-3-fold higher in estrogen receptor-positive (ERϩ) than in estrogen receptor-negative (ERϪ) breast tumors (7). The growth of breast cancer cells is highly sensitive to anti-calmodulin drugs (8, 9), and the anti-estrogen Tamoxifen binds to CaM with high affinity and antagonizes its action (10). CaM also modulates estrogen (17␤-estradiol (E2)) binding to ER (11), and synergistic inhibition of growth by CaM inhibitors and antiestrogens (12, 13) are associated with breast cancer.Both anti-estrogens and anti-calmodulin drugs block breast cancer cells at an identical point in the G 1 phase of the cell cycle (14). Furthermore, CaM is known to stimulate the phosphorylation of estrogen receptors (15). Biochemical evidence suggests an interaction of CaM with ER (11, 16 -19), but these results did not demonstrate a direct physical contact.We examined more directly the possibility of a role of CaM in key downstream events of E2 action, i.e. the interaction of ER protein with its cognate DNA sequence (estrogen response element (ERE)) to form the ER⅐ERE complex and the resulting transactivation of an E2-responsive promoter. We observed that CaM directly interacts with ER, and is not only an integral component of the ER⅐ERE complex but also plays an essential role in complex formation. Its functional involvement in the molecular pathway of E2-induced transactivation of hormoneresponsive genes in breast cancers was tested. EXPERIMENTAL PROCEDURESCell Lines and Culture-MCF-7 and MDA-MB-231 cell lines were obtained from ATCC and were grown in rich medium (Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2.8 M hydrocortisone, 1 g/ml insulin, and 12.5 ng/ml epide...

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Section: Cam Depletion From Nuclear Extract Of Mda-mb-231 Cells Prevementioning

confidence: 92%

“…Purified ER formed a complex with 32 P-labeled ERE oligonucleotide only in the presence of nuclear extract from MDA-MB-231 cells (Fig. 1, A, lane 3, and B, lane 5; each lane is represented by duplicate samples), suggesting that auxiliary proteins are necessary for ER⅐ERE complex formation.…”

Section: Cam Is An Integral Component Of the Er⅐ere Complex-wementioning

confidence: 98%

Calmodulin Is Essential for Estrogen Receptor Interaction with Its Motif and Activation of Responsive Promoter

Biswas¹,

Reddy²,

Pickard³

et al. 1998

Journal of Biological Chemistry

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“…CaM-affinity chromatography was performed essentially as described (34). Briefly, whole-cell lysate of LNCaP cells prepared as described above was diluted 20-fold in buffer B (20 mM Tris, pH 7.4͞4 mM MgCl 2 ͞2 mM CaCl 2 ͞10 mM KCl͞1 mM PMSF) and applied to a CaM-agarose (Sigma) affinity column.…”

Section: Methodsmentioning

confidence: 99%

Physical and functional interaction of androgen receptor with calmodulin in prostate cancer cells

Cifuentes

Mataraza

Yoshida

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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Ca 2؉ and calmodulin (CaM) play a critical role in proliferation and viability of a wide variety of cells, including prostate cancer cells. We examined two prostate cancer cell lines, androgen-sensitive LNCaP and androgen-independent PC-3. Proliferation of LNCaP cells was six to eight times more sensitive to the inhibitory effect of the CaM antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) than were PC-3 cells. Because LNCaP cell proliferation is sensitive to stimulation by androgen, we assessed the physical and functional interaction between androgen receptor (AR) and CaM. We observed tight binding of AR to CaM when LNCaP cell extracts were subjected to CaM-affinity column chromatography. AR binding to CaM was Ca 2؉ -dependent and was inhibited by pretreatment of the cell extracts with W-7. Using immunofluorescence staining and confocal microscopy, we demonstrated colocalization of AR and CaM in the nucleus of LNCaP cells. Furthermore, the functional relevance of AR-CaM interactions in intact cells was revealed by the observation that W-7 was as effective as Casodex, an antiandrogen, in blocking AR-regulated expression of prostate-specific antigen in LNCaP cells. AR seems to interact with CaM directly because purified human AR could bind to CaM-agarose, and CaM could be detected in AR-immunoprecipitate prepared from purified soluble proteins. These studies provide direct evidence for physical and functional interaction between AR and CaM and suggest the potential usefulness of CaM antagonists in blocking AR activity in prostate cancer.

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“…CaM (calmodulin), a ubiquitous Ca 2+ -binding protein belonging to the EF hand homologue family, regulates a whole host of processes within the cytoplasm and nucleus of all eukaryotic cells [1,2]. Within the nucleus CaM has been found to be involved in RNA synthesis [3], DNA synthesis [4][5][6][7] and the function of a number of nuclear enzymes and receptors including the oestrogen receptor-α [8,9]. Ca 2+ /CaM-dependent phosphorylation of three nuclear proteins of 50-60 kDa [10], of CREB (cAMP-responseelement-binding protein) [11] and of rat liver hnRNPs (heterogenous nuclear ribonucleoproteins) A2 and C has been reported previously [12].…”

Section: Introductionmentioning

confidence: 99%

Role of Ca2+ activation and bilobal structure of calmodulin in nuclear and nucleolar localization

Thorogate

Török

2007

Biochemical Journal

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Ca2+ signalling to the nucleus is thought to occur by calmodulin entry into the nucleus where calmodulin has many functions. In the present study we have investigated the role of Ca2+ and the N- and C-terminal lobes of calmodulin in its subnuclear targeting by using fluorescently labelled calmodulin and its mutants and confocal microscopy. Our data show, first, that Ca2+ stimulation induces a reorganization of subnuclear structures to which apo-calmodulin can bind. Secondly, Ca2+-independent association of the C-terminal lobe is seen with subnuclear structures such as chromatin, the nuclear envelope and the nucleoli. Thirdly, Ca2+-dependent accumulation of both calmodulin and the C-terminal calmodulin lobe occurs in the nucleoli. The N-terminal lobe of calmodulin does not show significant binding to subnuclear structures although, similarly to the C-terminal lobe, it accumulates in the nucleoplasm of wheat germ agglutinin-blocked nuclei suggesting that a facilitated nuclear export mechanism exists for calmodulin.

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Growth factor modulated calmodulin-binding protein stimulates nuclear DNA synthesis in hemopoietic progenitor cells

Cited by 14 publications

References 41 publications

Calmodulin Is Essential for Estrogen Receptor Interaction with Its Motif and Activation of Responsive Promoter

Calmodulin Is Essential for Estrogen Receptor Interaction with Its Motif and Activation of Responsive Promoter

Physical and functional interaction of androgen receptor with calmodulin in prostate cancer cells

Role of Ca2+ activation and bilobal structure of calmodulin in nuclear and nucleolar localization

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