Role of breast tumour kinase in the in vitro differentiation of HaCaT cells.
BACKGROUND
Breast tumour kinase (BRK) is a newly identified non-receptor protein tyrosine kinase from a metastatic breast tumour. Its biological functions are still under extensive investigation. The mouse homologue Sik (Src-related intestinal kinase) has been implicated in mouse keratinocyte differentiation; however, not much is known about the functions of BRK in human cutaneous biology.


OBJECTIVE
Using HaCaT cells as an experimental model, to explore the mutual relationships between BRK and differentiation of human keratinocytes.


METHODS
Archival paraffin blocks of normal and pathological skin were retrieved for examining the in vivo distribution of BRK. Its expression and subcellular localization were examined via indirect immunofluorescence, and quantitative changes were analysed by Northern and Western blots. The kinase activity of BRK was determined by its autophosphorylation and phosphorylation of exogenous substrate in the in vitro kinase assay. Using a retroviral infection method, we established stably transfected HaCaT cells expressing vector, wild-type BRK or a kinase-defective mutant (K219M). Expression of the differentiation marker keratin 10 (K10) was compared among these cells using semiquantitative reverse transcription-polymerase chain reaction. Results Histochemical examination showed that BRK was expressed exclusively in suprabasal keratinocytes. Its distribution was both cytoplasmic and intranuclear. An enhanced regional suprabasal expression pattern was observed in the confluent areas of cell cultures. The expression of BRK transcript and protein was up-regulated in prolonged confluence culture in a serum-dependent manner. Its kinase activity was activated shortly after the addition of calcium and ionomycin and returned to the basal level within 30 min. Overexpression of wild-type BRK moderately promoted the expression of K10 transcript while the kinase-defective BRK mutant exerted a prominent suppressive effect.


CONCLUSIONS
The in vivo distribution of BRK and its up-regulation during in vitro differentiation of HaCaT cells, together with the activation of its kinase activity by calcium/ionomycin and its influence on K10 expression, all indicate a role for BRK in the complex process of keratinocyte differentiation.
Wang
T C
TC
Department of Dermatology, College of Medicine, National Taiwan University and National Taiwan University Hospital, Taipei, Taiwan.
Jee
S H
SH
Tsai
T F
TF
Huang
Y L
YL
Tsai
W L
WL
Chen
R H
RH
D015153:Blotting, Western / Q000379:methods; D002118:Calcium / Q000494:pharmacology; D002454:Cell Differentiation / Q000502:physiology*; D045744:Cell Line, Tumor; D004817:Epidermis / Q000201:enzymology / Q000503:physiopathology; D019084:Fluorescent Antibody Technique, Indirect / Q000379:methods; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D053550:Keratin-10; D015603:Keratinocytes / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D007633:Keratins / Q000032:analysis; D009154:Mutation / Q000235:genetics; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation / Q000502:physiology
D007476:Ionophores; C507493:KRT10 protein, human; C507491:Krt10 protein, mouse; D009363:Neoplasm Proteins; D053550:Keratin-10; D015759:Ionomycin; D007633:Keratins; D011505:Protein-Tyrosine Kinases; C088375:PTK6 protein, human; D002118:Calcium