Nuclear localization of 68 kDa calmodulin‐binding protein is associated with the onset of DNA replication

Subramanyam, Chakrapani; Honn, Susan C.; Reed, W. C.; Reddy, G. Prem Veer

doi:10.1002/jcp.1041440309

Cited by 35 publications

(18 citation statements)

References 37 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%

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%

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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“…LNCaP cells were subjected to nuclear and cytoplasmic fractionation by slight modification of the method of Subramanyam et al (30). All chemicals were obtained from Sigma-Aldrich (St. Louis, MO), except where otherwise noted.…”

Section: Methodsmentioning

confidence: 99%

Calmodulin-Androgen Receptor (AR) Interaction: Calcium-Dependent, Calpain-Mediated Breakdown of AR in LNCaP Prostate Cancer Cells

Pelley¹,

Chinnakannu²,

Murthy³

et al. 2006

Cancer Research

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Chemotherapy of prostate cancer targets androgen receptor (AR) by androgen ablation or antiandrogens, but unfortunately, it is not curative. Our attack on prostate cancer envisions the proteolytic elimination of AR, which requires a fuller understanding of AR turnover. We showed previously that calmodulin (CaM) binds to AR with important consequences for AR stability and function. To examine the involvement of Ca 2+ /CaM in the proteolytic breakdown of AR, we analyzed LNCaP cell extracts that bind to a CaM affinity column for the presence of low molecular weight forms of AR (intact AR size, f114 kDa). Using an antibody directed against the NH 2 -terminal domain (ATD) of AR on Western blots, we identified f76-kDa, f50-kDa, and 34/31-kDa polypeptides in eluates of CaM affinity columns, suggesting the presence of CaM-binding sites within the 31/34-kDa ATD of AR. Under cell-free conditions in the presence of phenylmethylsulfonyl fluoride, AR underwent Ca 2+ -dependent degradation. AR degradation was inhibited by N-acetyl-leu-leu-norleu, an inhibitor of thiol proteases, suggesting the involvement of calpain. In intact cells, AR breakdown was accelerated by raising intracellular Ca 2+ using calcimycin, and increased AR breakdown was reversed with the cell-permeable Ca 2+ chelator bis-(O-aminophenoxy)-ethane-N,N,N ¶,N ¶-tetraacetic acid tetra-(acetoxymethyl)-ester. In CaM affinity chromatography studies, the Ca 2+ -dependent protease calpain was bound to and eluted from the CaM-agarose column along with AR. Caspase-3, which plays a role in AR turnover under stress conditions, did not bind to the CaM column and was present in the proenzyme form. Similarly, AR immunoprecipitates prepared from wholecell extracts of exponentially growing LNCaP cells contained both calpain and calpastatin. Nuclear levels of calpain and calpastatin (its endogenous inhibitor) changed in a reciprocal fashion as synchronized LNCaP cells progressed from G 1 to S phase. These reciprocal changes correlated with changes in AR level, which increased in late G 1 phase and decreased as S phase progressed. Taken together, these observations suggest potential involvement of AR-bound CaM in calciumcontrolled, calpain-mediated breakdown of AR in prostate cancer cells. (Cancer Res 2006; 66(24): 11754-62)

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“…Preparation of Nuclear and Cytoplasmic Fractions. Exponentially growing LNCaP and PC-3 cells were subjected to nuclear and cytoplasmic fractionation as described (33).…”

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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Nuclear localization of 68 kDa calmodulin‐binding protein is associated with the onset of DNA replication

Cited by 35 publications

References 37 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

Calmodulin-Androgen Receptor (AR) Interaction: Calcium-Dependent, Calpain-Mediated Breakdown of AR in LNCaP Prostate Cancer Cells

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

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