Interaction of calmodulin with a putative calmodulin‐binding domain of inositol 1,4,5‐triphosphate 3‐kinase.

Abstract

Recombinant rat brain inositol 1,4,5‐triphosphate [Ins(1,4,5)P3] 3‐kinase was expressed in Escherichia coli as a β‐galactosidase fusion product. It could be adsorbed onto calmodulin‐Sepharose and eluted in Ca2+‐free medium as a 48‐kDa protein. Purification could be achieved in a single step. Molecular evidence for a calmodulin‐binding domain on Ins(1,4,5)P3 3‐kinase can be shown by the following approaches. (a) Inhibition of Ca2+/calmodulin stimulation by a synthetic peptide based on a candidate calmodulin‐binding domain. The inhibition was mimicked by a well‐characterized peptide derived from the sequence of smooth muscle myosin light‐chain kinase calmodulin‐binding site. (b) The construction of two mutants by site‐directed mutagenesis of Trp165 to Gly or Arg. Both mutants displayed kinase activity but were no longer Ca2+/calmodulin sensitive, supporting, therefore, the role of Trp165 in calmodulin binding.

Regulation of Inositol 1,4,5-Trisphosphate 3-Kinases by Calcium and Localization in Cells

“…5C), as previously demonstrated for IP3KA (46). It is interesting to note in passing that the expressed IP 3 KB in particular shows evidence of being proteolyzed, much more so than the other isoforms, consistent with it being particularly sensitive to proteolysis in vitro (44) and in vivo (25); we have shown that its subcellular localization is altered by that proteolysis (25).…”

“…5A), highlighting in particular the essential tryptophan residue (arrow) that is highly conserved and crucial to CaM binding (46). The C. elegans enzyme is known not to bind CaM (18), and this is consistent with the absence of this tryptophan in the C. elegans sequence.…”

“…To test CaM binding from bacterial lysates, we adopted a published procedure (46). 10 l of crude lysate was diluted in 800 l of binding buffer (20 mM Tris, pH 7.5, 0.2 mM CaCl 2 , 0.1 M NaCl, 1% Triton X-100, 1% fish skin gelatin) and applied to a 50-l 1:1 slurry of pre-equilibrated CaM-agarose resin (Calbiochem).…”

Integrated Protein Array Screening and High Throughput Validation of 70 Novel Neural Calmodulin-binding Proteins

“…For example, inositol 1,4,5-trisphosphate is a major trigger of Ca 2ϩ release in the cell though binding to the inositol 1,4,5-trisphosphate receptor, which in turn is regulated by calmodulin (49). Calmodulin also binds to and regulates phosphatidylinositol 3-kinase and inositol-1,4,5-trisphosphate 3-kinase (50). Additional links are provided by calbindin D28k, which activates myo-inositol-1(or 4)-monophosphatase (51), and frequenin (neuronal calcium sensor-1), which modulates the activity of phosphatidylinositol 4-kinase (52).…”

Inositol 1,4,5-trisphosphate 3-kinases: functions and regulations

“…CaM recognizes sequences which contain amphiphilic α-helices with clusters of positively charged and hydrophobic amino acids [38]. Sequence from Ser-156 to Leu-189 together with site Trp-165 in rat IP 3 3K-A is required for CaM binding and the enzyme activation [38,48,49]. The level of stimulation appears to be cell-, tissue-and isoform-specific [27,50] (Tab.…”