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dictyNews Volume 34 Number 06
dictyNews
Electronic Edition
Volume 34, number 6
February 19, 2010
Please submit abstracts of your papers as soon as they have been
accepted for publication by sending them to dicty@northwestern.edu
or by using the form at
http://dictybase.org/db/cgi-bin/dictyBase/abstract_submit.
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Abstracts
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Galpha5 subunit-mediated signaling requires a D-motif and the MAP kinase
ERK1 in Dictyostelium
Brent Raisley1, Hoai-Nghia Nguyen, and Jeffrey A. Hadwiger*
Department of Microbiology and Molecular Genetics, Oklahoma State University,
Stillwater, Oklahoma 74078-3020, USA
Microbiology, in press
The Dictyostelium Galpha5 subunit has been shown to reduce cell viability, inhibit
folate chemotaxis and to accelerate tip morphogenesis and gene expression
during multicellular development. Alteration of the D-motif [mitogen-activated
protein kinase (MAPK) docking site] at the amino terminus of the Galpha5 subunit
or the loss of the MAPK ERK1 diminished the lethality associated with the
over-expression or constitutive activation of the Galpha5 subunit. The amino
terminal D-motif of the Galpha5 subunit was also found necessary for the reduced
cell size, small aggregate formation and precocious developmental gene
expression associated with Galpha5 subunit over-expression. This D-motif also
contributed to the aggregation delay in cells expressing a constitutively-active
Galpha5 subunit but the D-motif was not necessary for the inhibition of folate
chemotaxis. These results suggest that the amino terminal D-motif is required
for some but not all phenotypes associated with elevated Galpha5 subunit
functions during growth and development and that ERK1 can function in Galpha5
subunit-mediated signal transduction.
Submitted by Jeff Hadwiger [jeff.hadwiger@okstate.edu]
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MAP Kinases have different functions in Dictyostelium
G Protein-Mediated Signaling
Hoai-Nghia Nguyen, Brent Raisley1, and Jeffrey A. Hadwiger*
Department of Microbiology and Molecular Genetics, Oklahoma State University,
Stillwater, Oklahoma 74078-3020, USA
Cellular Signalling, in press
Extracellular signal regulated kinases (ERKs) are a class of MAP kinases that
function in many signaling pathways in eukaryotic cells and in some cases, a
single stimulus can activate more than one ERK suggesting functional
redundancy or divergence from a common pathway. Dictyostelium discoideum
encodes only two MAP kinases, ERK1 and ERK2, that both function during the
developmental life cycle. To determine if ERK1 and ERK2 have overlapping
functions, chemotactic and developmental phenotypes of erk1- and erk2-
mutants were assessed with respect to G protein-mediated signal transduction
pathways. ERK1 was specifically required for Gα5-mediated tip
morphogenesis and inhibition of folate chemotaxis but not for cAMP-stimulated
chemotaxis or cGMP accumulation. ERK2 was the primary MAPK
phosphorylated in response to folate or cAMP stimulation. Cell growth was
not altered in erk1-, erk2- or erk1-erk2- mutants but each mutant displayed a
different pattern of cell sorting in chimeric aggregates. The distribution of
GFP-ERK1 or GFP-ERK2 fusion proteins in the cytoplasm and nucleus was
not grossly altered in cells stimulated with cAMP or folate. These results suggest
ERK1 and ERK2 have different roles in G protein-mediated signaling during
growth and development.
Submitted by Jeff Hadwiger [jeff.hadwiger@okstate.edu]
--------------------------------------------------------------------------------
Mutation of Actin Tyr-53 Alters the Conformations of the DNase I-binding
Loop and the Nucleotide-binding Cleft
Xiong Liu, Shi Shu, Myoung-Soon S. Hong, Bin Yu and, Edward D. Korn
From the Laboratory of Cell Biology, National Heart, Lung, and Blood Institute,
NIH, Bethesda, MD 20892
J. Biol. Chem. , in press
All but 11 of the 323 known actin sequences have Tyr at position 53, and the 11 exceptions
have the conservative substitution Phe, which raises the questions: What is(are) the critical
role(s) of Tyr-53, and, if it can be replaced by Phe, why has this happened so infrequently?
We compared the properties of purified endogenous Dictyostelium actin and mutant
constructs with Tyr-53 replaced by Phe, Ala, Glu, Trp and Leu. The Tyr53Phe mutant did
not differ significantly from endogenous actin in any of the properties assayed, but the
Tyr53Ala and Tyr53Glu mutants differed substantially: affinity for DNase I was
reduced, the rate of nucleotide exchange was increased, the critical concentration for
polymerization was increased, filament elongation was inhibited, and polymerized
actin was in the form of small oligomers and imperfect filaments. Growth and/or
development of cells expressing these actin mutants were also inhibited. The Trp and
Leu mutations had less, but still significant, effects. We conclude that either Tyr or Phe is
required to maintain the functional conformations of the DNase I-binding loop
(D-loop), and that the conformation of the D-loop affects not only the properties that
directly involve the D-loop but also allosterically modifies the conformation of the
nucleotide-binding cleft. The apparent evolutionary “preference” for Tyr at position
53 may be because Tyr allows dynamic modification of the D-loop conformation by
phosphorylation (Baek et al. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 11748-11753)
with effects similar, but not identical, to those of the Ala and Glu mutations.
Submitted by Edward Korn [korned2@nhlbi.nih.gov]
--------------------------------------------------------------------------------
Identification of a new mechanism for targeting myosin II heavy chain
phosphorylation by Dictyostelium myosin heavy chain kinase B
Julie Underwood, Jonathan Greene, and Paul A. Steimle
Department of Biology, University of North Carolina at Greensboro,
Greensboro, North Carolina, USA
BMC Research Notes, in press
BACKGROUND: Heavy chain phosphorylation plays a central role in regulating
myosin II bipolar filament assembly in Dictyostelium, as well as in higher
eukaryotic nonmuscle cells. Our previous work has demonstrated that the
WD-repeat domain of Dictyostelium myosin II heavy chain kinase B (MHCK-B),
unlike its counterpart in MHCK-A, is not absolutely required for targeting of the
kinase to Phosphorylate MHC. Thus, we tested the hypothesis that an
asparagine-rich and structurally disordered region that is unique to MHCK-B
can by itself function in substrate targeting.
FINDINGS: Biochemical assays comparing the activities of full-length MHCK-B,
a truncation lacking only the WD-repeat domain (B-delta-WD), and a truncation
lacking both the N-rich region and the WD-repeat domain (B-delta-N-WD) revealed
that the N-rich region targets MHCK-B to phosphorylate MHC in a manner that
leads to bipolar filament disassembly. This targeting is physiologically relevant
since cellular over-expression of the B-delta-WD truncation, but not the
B-delta-N-WD truncation, leads to dramatically reduced levels of myosin II
filament assembly and associated defects in cytokinesis and multicellular
development.
CONCLUSIONS: The results presented here demonstrate that an intrinsically
unstructured, and asparagine-rich, region of a MHCK-B can mediate specific
targeting of the kinase to phosphorylate myosin II heavy chain. This targeting
involves a direct binding interaction with myosin II filaments. In terms of
regulating myosin bipolar filament assembly, our results suggest that factors
affecting the activity of this unique region of MHCK-B could allow for
regulation of MHCKB in a manner that is distinct from the other MHCKs
in Dictyostelium.
Submitted by Paul Steimle [p_steiml@uncg.edu]
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[End dictyNews, volume 34, number 6]
