Identification of two phosphorylated threonines in the tail region of Dictyostelium myosin II.

145

152

We have identified a Dictyostelium discoideum gene encoding a serine/threonine kinase, PAKa, a putative member of the Ste20/PAK family of p21-activated kinases, with a kinase domain and a long NH2-terminal regulatory domain containing an acidic segment, a polyproline domain, and a CRIB domain. PAKa colocalizes with myosin II to the cleavage furrow of dividing cells and the posterior of polarized, chemotaxing cells via its NH2-terminal domain. paka null cells are defective in completing cytokinesis in suspension. PAKa is also required for maintaining the direction of cell movement, suppressing lateral pseudopod extension, and proper retraction of the posterior of chemotaxing cells. paka null cells are defective in myosin II assembly, as the myosin II cap in the posterior of chemotaxing cells and myosin II assembly into cytoskeleton upon cAMP stimulation are absent in these cells, while constitutively active PAKa leads to an upregulation of myosin II assembly. PAKa kinase activity against histone 2B is transiently stimulated and PAKa incorporates into the cytoskeleton with kinetics similar to those of myosin II assembly in response to chemoattractant signaling. However, PAKa does not phosphorylate myosin II. We suggest that PAKa is a major regulator of myosin II assembly, but does so by negatively regulating myosin II heavy chain kinase.

Section: Possible Model For Paka Function In Controlling Myosin II Assemblymentioning

confidence: 99%

Paka, a Putative Pak Family Member, Is Required for Cytokinesis and the Regulation of the Cytoskeleton in Dictyostelium discoideum Cells during Chemotaxis

Chung

Firtel

1999

145

152

“…Recently, Egelhoff et al (7) have transformed myosin null Dictyostelium cells with a vector expressing an altered myosin H gene that eliminates the terminal 34-kD of the tail of myosin H heavy chain (the site of myosin H heavy chain phosphorylation) (29). These transformants display excessive localization of truncated myosin H in the cortical cytoskeleton.…”

Section: Phosphorylation Of Myosin H Heavy Chains Is Prerequisite To the Release Of Myosin H From Membrane-cytoskeletonsmentioning

confidence: 99%

Release of myosin II from the membrane-cytoskeleton of Dictyostelium discoideum mediated by heavy-chain phosphorylation at the foci within the cortical actin network

Yumura

Kitanishi-Yumura

1992

Abstract. Membrane-cytoskeletons were prepared from Dictyostelium amebas, and networks of actin and myosin II filaments were visualized on the exposed cytoplasmic surfaces of the cell membranes by fluorescence staining (Yumura, S., and T. . Cell . Addition of ATP caused contraction of the cytoskeleton with aggregation of part of actin into several foci within the network, but most of myosin II was released via the foci. However, in the presence of 10 mM MgCI2, which stabilized myosin II filaments, myosin II remained at the foci. Uttrastructural examination revealed that, after contraction, only traces of monomeric myosin II remained at the foci. By contrast, myosin H filaments remained in the foci in the presence of 10 mM MgCI2. These observations suggest that myosin II was released not in a filamentous form but in a monomeric form.Using [732P]ATP, we found that the heavy chains of myosin II released from membrane-cytoskeletons were phosphorylated, and this phosphorylation resulted in disassembly of myosin filaments. Using ITP (a substrate for myosin 1I ATPase) and/or ATP3tS (a substrate for myosin II heavy-chain kinase [MHCK]), we demonstrated that phosphorylation of myosin heavy chains occurred at the foci within the actin network, a result that suggests that MHCK was localized at the loci. These results together indicate that, during contraction, the heavy chains of myosin II that have moved toward the foci within the actin network are phosphorylated by a specific MHCK, with the resultant disassembly of filaments which are finally released from membrane-cytoskeletons. This series of reactions could represent the mechanism for the relocation of myosin II from the cortical region to the endoplasm. MYOSIS II, which is one of the major components of the cytoskeleton in nonmuscle cells, produces the motive force necessary for cell movements and cytokinesis via interactions with actin filaments. Myosin II isolated from Dictyostelium amebas can assemble into bipolar thick filaments in vitro (28). Immunofluorescence studies and irm'nunoelectron microscopy have shown that myosin II in Dictyostelium amebas, similar to myosin in muscle cells, forms filaments in vivo (30, 31). In addition, most of the myosin II in Triton-Xl00-insoluble cytoskeletons of Dictyostelium amebas is in the filamentous form (4, 27). All these observations suggest that, in Dictyostelium aniebas, actin and myosin II generate the motive force by a mechanism analogous to the sliding-filament model of actomyosin in muscle cells. However, actin and myosin filaments in D/ctyostelium amebas, unlike those in muscle cells, show no evidence of any regular arrangement such as that observed in the sarcomere in muscle cells and, in addition, they do not stay at a single site but can relocate within a cell. For example, myosin H filaments are concentrated at the tail region during locomotion, while they are concentrated in the furrow region to form the contractile ring during cell division (12,30,33). Upon chemotactic stimulation of cells at the aggregation st...

“…The distribution of phosphates among the heavy chains is not homogeneous since only 43 % of the molecules are bent monomers when 1 mol of 32p is incorporated per mole of myosin heavy chain. Several sites on the heavy chain may be phosphorylated (Vaillancourt et al, 1988;Ravid and Spudich, 1989).…”

Section: Phosphorylation Of the Heavy Chain Correlates With Bending Of The Myosin Tailmentioning

confidence: 99%

Intermolecular versus intramolecular interactions of Dictyostelium myosin: possible regulation by heavy chain phosphorylation.

Pasternak

Flicker

Ravid

et al. 1989