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Phosphorylated HSP27 associates with the activation-dependent cytoskeleton
in human platelets
Y Zhu, S O'Neill, J Saklatvala, L Tassi and ME Mendelsohn
Molecular Cardiology Research Center, New England Medical Center, Tufts
University School of Medicine, Boston, MA 02111.
The three prominently phosphorylated 29-kD proteins in thrombin- activated
human platelets are forms of the mammalian 27-kD heat-shock protein
(HSP27). Though the function of HSP27 is not yet known, its phosphorylation
is highly correlated with platelet secretion, and recent evidence in
nonhematopoietic cells suggests that HSP27 regulates cortical actin
filament assembly. Therefore, the subcellular location and phosphorylation
state of HSP27 in resting and thrombin-activated platelets was studied.
Platelets were fractionated by established Triton X-100 lysis methods
followed by differential centrifugation to obtain the 14,000g fraction
(low-speed cytoskeleton), 100,000g fraction (membrane skeleton), and the
100,000g supernatant fraction containing soluble cytosolic proteins. In
resting platelets, HSP27 was present principally in the 100,000g
supernatant fraction. Platelet activation with thrombin led to
translocation of the majority of HSP27 to the low- speed cytoskeleton. This
association was reversible by DNase, supporting the idea that HSP27 is a
specific component of the actin cytoskeleton. Immunofluorescence studies
similarly showed HSP27 is cytoplasmic in resting platelets but colocalizes
with actin in fully spread, glass-activated platelets. Immunoprecipitation
studies showed a small amount of constitutively phosphorylated HSP27 in
resting platelets, but phosphorylation of the majority of HSP27 after
thrombin activation. After activation, virtually all phosphorylated HSP27
was found in the low-speed cytoskeletal fraction, and each of the three
phosphorylated forms of HSP27 were present by two-dimensional
autoradiography. Furthermore, in time-course studies, the phosphorylation
of HSP27 occurred just before localization of HSP27 to the low-speed
pellet. These results show that, after platelet activation, HSP27 is first
phosphorylated and then translocated from the cytoplasm to the assembling
cytoskeleton, and suggest that HSP27 phosphorylation may be important to
the binding of HSP27 to cytoskeletal components and the cytoskeletal
rearrangements characteristic of platelet activation.
Volume 84,
Issue 11,
pp. 3715-3723,
12/01/1994
Copyright © 1994 by The American Society of Hematology
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