Recruitment of the LIM protein hic-5 to focal contacts of human platelets.
Platelets are anuclear, membrane-bounded fragments derived from megakaryocytes which, upon stimulation, assemble an actin skeleton including stress fibres and focal contacts. The focal contacts resemble those of tissue culture cells. However, they lack paxillin, a conspicuous component of these organelles. We found that instead of paxillin, platelets contain a related protein with a molecular mass of 55 kDa that crossreacts with a monoclonal antibody against paxillin. The gene for the 55 kDa protein was cloned from a bone marrow cDNA library and turned out to be identical to a recently discovered gene encoding hic-5. Like paxillin, hic-5 is a cytoskeletal protein containing four carboxy-terminal LIM domains and LD motifs in the amino-terminal half. The LIM domains of both hic-5 and paxillin are capable of targetting green fluorescent protein to focal contacts. In addition, GST-hic-5 precipitates the focal adhesion kinase pp125(FAK) and talin from platelet extracts. Only trace amounts of hic-5 occur in DAMI cells, a megakaryocytic cell line, and in megakaryocytes cultured from CD34+ cells obtained from umbilical cord blood. However, RT-polymerase chain reactions performed with RNA obtained from platelets gave a positive result when primers specific for hic-5 were used, but were negative with paxillin-specific primers, indicating that a switch from paxillin expression to hic-5 expression must occur late in the maturation of megakaryocytes into platelets.
Friedrich Miescher-Institute, Basel, Switzerland.
J
J
Hagmann
M
M
Grob
A
A
Welman
G
G
van Willigen
M M
MM
Burger
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001792:Blood Platelets / Q000166:cytology*; D001853:Bone Marrow; D002448:Cell Adhesion / Q000502:physiology*; D015815:Cell Adhesion Molecules / Q000032:analysis; D002460:Cell Line; D003001:Cloning, Molecular; D003429:Cross Reactions; D003598:Cytoskeletal Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D005312:Fetal Blood; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D060588:LIM Domain Proteins; D008533:Megakaryocytes / Q000737:chemistry; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D051419:Paxillin; D010750:Phosphoproteins / Q000032:analysis / Q000235:genetics; D011505:Protein-Tyrosine Kinases / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D011993:Recombinant Fusion Proteins; D016608:Talin / Q000032:analysis
D015815:Cell Adhesion Molecules; D003598:Cytoskeletal Proteins; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D060588:LIM Domain Proteins; C496061:PXN protein, human; D051419:Paxillin; D010750:Phosphoproteins; C496062:Pxn protein, mouse; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C490351:TGFB1I1 protein, human; D016608:Talin; C090090:Tgfb1i1 protein, mouse; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; C496051:Ptk2 protein, mouse