Biochemical evidence for a calmodulin‐stimulated calcium‐dependent protein kinase in maize

Abstract

We provide biochemical evidence for the presence of a Ca 2ϩ -dependent calmodulin (CaM)-stimulated protein kinase (CCaMK) from etiolated maize coleoptiles. The kinase, with a molecular mass of 72.3 kDa, was purified to homogeneity by means of ammonium sulphate precipitation, DEAE-Sephacel chromatography, CaM-Sepharose chromatography and gel purification. The purified kinase required 5 mM Mg 2ϩ for activity and had an optimum pH of 7.5. The kinase is a Ca 2ϩ -binding protein, as was evident by 45 Ca 2ϩ -binding and Ca 2ϩ mobility-gel-shift assays. 1 µM Ca 2ϩ stimulated the kinase activity about 12-fold and was further stimulated by the addition of exogenous CaM (Ϸ100 nM). Addition of Ca 2ϩ and CaM antagonists decreased the kinase activity. Under in vitro assay conditions the kinase phosphorylated preferentially syntide-2, histone IIIS and casein. Syntide-2 and histone IIIS were phosphorylated at serine residues, showing that the kinase belongs to the serine/threonine family of protein kinases. Autophosphorylation of CCaMK occurred on threonine residue(s) and was Ca 2ϩ dependent. Addition of exogenous CaM had no effect on autophosphorylation. The properties of the maize kinase suggests that it is a CCaMK that shows dual stimulation with Ca 2ϩ and CaM for substrate phosphorylation and only Ca 2ϩ requirement for autophosphorylation. Antibodies raised against the kinase cross-reacted with maize total proteins to give a single band of 72 kDa and precipitated substrate (syntide-2 and histone IIIS)-phosphorylation and autophosphorylation activities in a specific manner. Localisation studies with antibodies showed that the kinase is ubiquitous.Keywords : calmodulin; Ca 2ϩ /calmodulin-dependent kinase ; Zea mays; protein kinase. Ca 2ϩ are one of the most common second messengers, and couple a large number of diverse stimuli to their characteristic responses in a very specific and precise manner in plant and animal systems [1Ϫ3]. In animal systems, the role of Ca 2ϩ as a second messenger has been very well established. Changes in intracellular Ca 2ϩ levels are perceived by various Ca 2ϩ -binding proteins, e.g. calmodulin (CaM) and CaM-like proteins, which either by themselves or by modifying other proteins and/or factors transduce the signal downstream to elicit the final response. Protein kinases are one of the main targets of these proteins, which by their reversible phosphorylation/dephosphorylation activate other constituents of signal-transduction pathways [1, 4Ϫ5].Ca 2ϩ is known to regulate two families of protein kinases, i.e. Ca 2ϩ /phospholipid-dependent protein kinases [6] and Ca 2ϩ / CaM-dependent protein kinases (CCaMK) [7Ϫ9]. In animal systems, various members of these families have been purified, and characterised, and their regulation has been understood [10]. In plants a third, biochemically distinct family of protein kinases, Ca 2ϩ -dependent protein kinases, is more common [11]. These kinases do not require lipid or CaM for their activation. These kinases are single polypeptides, containing conserved cata...

The Arabidopsis CDPK-SnRK Superfamily of Protein Kinases

“…The arrangement of functional domains in these enzymes is similar to that of the CDPKs, but the structure of their calciumbinding regulatory domain is more similar to visinin than to calmodulin and contains only three EF hands (Harmon et al, 1987;Patil et al, 1995;Yuasa et al, 1995;Pandey and Sopory, 1998). CCaMKs contain a calmodulin-binding site that overlaps with the autoregulatory domain (Ramachandiran et al, 1997).…”

“…Regulation of CCaMK by calcium and calmodulin is complex. Autophosphorylation occurs in response to the binding of calcium to the EF hands in the visininlike domain, which increases the affinity of the enzyme for binding the calcium/calmodulin complex and enhances substrate phosphorylation (Takezawa et al, 1996;Pandey and Sopory, 1998;Sathyanarayanan et al, 2000).…”

Flowers and mycorrhizal roots – closer than we think?

“…The precedent: the case of CCaMK CCaMK was first isolated from lilly (Lilium longiflorum) anthers [17], and subsequently biochemically characterized in lilly, tobacco (Nicotiana tabacum), maize (Zea mays), and pea (Pisum sativum) [18][19][20][21][22][23][24][25]. The combination of a calmodulin-binding domain and three EF hands in CCaMK is unique (Box 1) and sets it apart from the calcium-dependent protein kinases (CDPKs) [6].…”

Manipulação de cereais para acúmulo de lisina em sementes

“…O Ca 2+ é conhecido por modular a atividade de várias enzimas (Pandey & Sopory, 1998) sendo também um importante mensageiro em transdução de sinal em plantas (Snedden & Fromm, 1998). A calmodulina é um importante componente na regulação de complexos de quinases interagindo com vários tipos de moléculas efetoras (Soderling, 1999).…”