Molecular characterization of protein kinase C-alpha binding to lamin A.
Previous results from our laboratory have identified lamin A as a protein kinase C (PKC)-binding protein. Here, we have identified the regions of PKC-alpha that are crucial for this binding. By means of overlay assays and fusion proteins made of glutathione-S-transferase (GST) fused to elements of rat PKC-alpha, we have established that binding occurs through both the V5 region and a portion of the C2 region (i.e., the calcium-dependent lipid binding (CaLB) domain) of the kinase. In particular, we have found that amino acid 200-217 of the CaLB domain are essential for binding lamin A, as a synthetic peptide corresponding to this stretch of amino acids prevented the interaction between the CaLB domain and lamin A. We also show that the presence of four lysine residues of the CaLB domain (K205, K209, K211, and K213) was essential for the binding. We have determined that binding of elements of PKC-alpha to lamin A does not require the presence of cofactors such as phosphatidylserine (PS) and Ca(2+). We have also found that the binding site of lamin A for the CaLB domain of PKC-alpha is localized in the carboxyl-terminus of the lamin, downstream of amino acid 499. Our findings may prove to be important to clarify the mechanisms regulating PKC function within the nucleus and may also lead to the synthesis of isozyme-specific drugs to attenuate or reverse PKC-dependent nuclear signaling pathways important for the pathogenesis of cancer.
Dipartimento di Scienze Anatomiche Umane e Fisiopatologia dell'Apparato Locomotore, Sezione di Anatomia Umana, Cell Signaling Laboratory, Università di Bologna, via Irnerio 48, Italy. amartell@biocfarm.unibo.it
Alberto M
AM
Martelli
Roberta
R
Bortul
Giovanna
G
Tabellini
Irene
I
Faenza
Alessandra
A
Cappellini
Renato
R
Bareggi
Lucia
L
Manzoli
Lucio
L
Cocco
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D034904:Lamin Type A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011493:Protein Kinase C / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D007527:Isoenzymes; D034904:Lamin Type A; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha