Identification and Characterization of a Novel α-Kinase with a von Willebrand Factor A-like Motif Localized to the Contractile Vacuole and Golgi Complex inDictyostelium discoideum

Abstract

We have identified a new protein kinase in Dictyostelium discoideum that carries the same conserved class of "␣-kinase" catalytic domain as reported previously in myosin heavy chain kinases (MHCKs) in this amoeba but that has a completely novel domain organization. The protein contains an N-terminal von Willebrand factor A (vWFA)-like motif and is therefore named VwkA. Manipulation of VwkA expression level via high copy number plasmids (VwkA ؉؉ cells) or gene disruption (vwkA null cells) results in an array of cellular defects, including impaired growth and multinucleation in suspension culture, impaired development, and alterations in myosin II abundance and assembly. Despite sequence similarity to MHCKs, the purified protein failed to phosphorylate myosin II in vitro. Autophosphorylation activity, however, was enhanced by calcium/calmodulin, and the enzyme can be precipitated from cellular lysates with calmodulin-agarose, suggesting that VwkA may directly bind calmodulin. VwkA is cytosolic in distribution but enriched on the membranes of the contractile vacuole and Golgi-like structures in the cell. We propose that VwkA likely acts indirectly to influence myosin II abundance and assembly behavior and possibly has broader roles than previously characterized ␣ kinases in this organism, which all seem to be MHCKs. INTRODUCTIONNearly all aspects of cell life are regulated by protein phosphorylation involving a large number of biochemical reactions and distinct signaling pathways. Existence of ϳ500 genes encoding various protein kinases in the human genome further underscores their importance in cell life (Manning et al., 2002). Most of these conventional protein kinases belong to serine/threonine/tyrosine, a kinase superfamily whose members carry a conserved catalytic domain with recognizable sequence similarity in spite of having different targets in the cell (Hardie, 1994;Hanks and Hunter, 1995). In recent years, however, biochemical and molecular approaches have led to the identification of more divergent classes of eukaryotic protein kinases carrying a catalytic domain sequence with no sequence similarity with conventional protein kinase catalytic domains (Manning et al., 2002). One major class of unconventional protein kinases was first identified via representatives of myosin heavy chain kinases (MHCKs) in Dictyostelium discoideum (Futey et al., 1995;Côté et al., 1997) and via identification of mammalian eEF-2 kinase (eEF-2K) as an unconventional protein kinase (Ryazanov et al., 1997). This novel, conserved group of unconventional eukaryotic protein kinases has been termed the ␣-kinase family (Ryazanov et al., 1999;Drennan and Ryazanov, 2004).Molecular cloning and biochemical characterization studies demonstrated that at least three Dictyostelium ␣-kinases genes encode enzymes that can specifically phosphorylate myosin II heavy chain (MHC) in vitro and in vivo, and were thus named as MHC kinase A (MHCK A), MHC kinase B (MHCK B) and MHC kinase C (MHCK C) (Futey et al., 1995;Clancy et al., 1997;Luo et al., 200...

α-Kinase 1, a New Component in Apical ProteinTransport

“…This new family of protein kinases was named ␣-kinase, based on the evidence that these protein kinases phosphorylate amino acids located within ␣-helices. Interestingly, a very recent work describes the identification of a new ␣-kinase in Golgi-like structures from Dictyostelium, which is, like ALPK1, characterized by a catalytic domain located at the C terminus of the polypeptide (40). Furthermore, TRPM7/ChK1 channel kinase, another member of this family, has recently been shown to phosphorylate annexin 1, a cytosolic linker molecule involved in protein transport (41).…”

Multiple Myosin II Heavy Chain Kinases: Roles in Filament Assembly Control and Proper Cytokinesis inDictyostelium

“…A short DNA sequence of 466 base pairs length was PCR amplified using MHCKE934 (5Ј-GCAGGTACCGGTAGAATGCCATCAACTGGC-3Ј) and MKLA1386 (5Ј-GCATCTAGACAATTGATATGCATTTCTAAGTGCACC-3Ј) from the N-terminus of mhkD gene. The purified DNA fragment was cloned as KpnI and XbaI fragment in pBsr-Nsi plasmid vector (Betapudi et al, 2005) to generate pBsr-Nsi-5Ј-MHCKD vector. Similarly, another DNA fragment of 526 base pairs length was PCR amplified using MHCKE2236 (5Ј-CAGATCCAAGCT-TCAGCATCTGCTGATGGTTACG-3Ј) and MHCKE2762 (5Ј-ACTAGAG-GCGCCCCAAACATACGTAATGATGTAATTGG-3Ј) primers from the C terminus of mhkD gene.…”

“…We have named this fifth alpha kinase VwkA. Biochemical analysis and cellular studies indicate that VwkA does not function as a direct MHC kinase (Betapudi et al, 2005). The sixth alpha kinase gene has been provisionally named AK1 (alpha kinase 1) during genome annotation.…”

Autophosphorylation Activates Dictyostelium Myosin II Heavy Chain Kinase A by Providing a Ligand for an Allosteric Binding Site in the α-Kinase Domain

“…The other two ␣-kinases, AK1 and VwkA, have domain structures unrelated to MHCK A. AK1 contains an Arf GTPase-activating protein domain and VwkA contains an N-terminal von Willebrand factor A-like domain. The function of AK1 is not known, whereas VwkA is involved in the regulation of contractile vacuole function (16,17). Mammals express six multidomain proteins with ␣-kinase domains (12).…”